JP2013190680A - Microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a microscope that enables a wide range of focus off-set without sacrificing a focusing accuracy with an inexpensive configuration.SOLUTION: The microscope comprises: a focusing motor driving unit that changes a relative distance between a specimen and an objective lens; an observation optical system that is an infinity corrected optical system for observing observation light outgoing from the specimen and passing through the objective lens; a focusing detection optical system that projects AF light with a different wavelength from illumination light and the observation light to the specimen through the objective lens, and condenses the AF light reflected on an interface between a glass bottom dish and the specimen and passing through the objective lens again; and a control unit that controls the focusing motor driving unit such that an observing position on the specimen set on the basis of a signal input via a jog encoder coincides with a focal position of the objective lens. The focusing detection optical system includes a condenser lens that shifts a condensing position of the AF light projected on the specimen closer to the objective lens side than the focal position of the objective lens.

Description

  The present invention relates to a microscope capable of automatically adjusting the focus position of an observation sample.

  In recent years, a microscope apparatus that can generate an observation image of a fine sample and display or record it as a video image has been widely used in research in biological fields, inspection processes in industrial fields, and the like.

  When observing a sample using a microscope apparatus, it is necessary to focus accurately. Further, in routine work such as an inspection process, it is very important to quickly focus in order to shorten the inspection time.

The focusing operation in the microscope apparatus is usually performed by operating a focusing handle provided in the microscope apparatus to adjust the focus of the objective lens with respect to the observation sample. However, when the focusing operation is performed manually, the user needs to master to some extent until the user can quickly focus. In particular, when the focal depth is shallow and the focusing range is narrow as in a high-magnification objective lens, considerable skill is required.
On the other hand, in the case of a microscope apparatus with poor operability in the focusing operation, there may be effects such as user fatigue and a reduction in production efficiency.

For this reason, many proposals have been made on a microscope apparatus having an automatic focusing function for automatically focusing.
For example, Patent Document 1 uses a two-divided detector as a focus detection light acquisition unit, corrects the wavelength difference between the infrared light for focus detection that differs for each objective lens and the visible light that is actually observed, An automatic lens that adjusts the focal position of infrared light by providing a lens that can move by a fixed amount in the optical axis direction so that a focusing operation (focus offset) can be performed at any position in the optical axis direction of the sample. A focus detection microscope is disclosed.

  Patent Document 2 discloses an automatic focus detection microscope capable of correcting chromatic aberration and focus offset using an area sensor such as a PSD (light receiving element) as focus detection light acquisition means.

Japanese Patent Laid-Open No. 11-249027 Japanese Patent Laid-Open No. 62-131219

  However, in the autofocus detection microscope disclosed in Patent Document 1, since a moving mechanism is provided in the focus detection optical system, the apparatus is increased in size and cost.

  On the other hand, in Patent Document 2, in order to ensure a wide focus offset amount in terms of optical characteristics, it is possible to capture a further change in the spot image by increasing the size of the light receiving surface of the area sensor, or It is necessary to reduce the sensitivity of spot image change by lowering the effective numerical aperture on the specimen side of autofocus (AF) light. However, increasing the size of the light receiving surface of the area sensor naturally increases the cost. Even if the sensor can be increased in size, in a region where the amount of focus offset is large, the area of the spot image increases and the light intensity per unit area decreases, so that a sufficient S / N can be obtained. Disappear. On the other hand, when the effective numerical aperture on the specimen side of the AF light is lowered, the focus offset amount is increased, but the focusing accuracy is lowered.

  The present invention has been made in view of the above, and an object thereof is to provide a microscope that can be realized with an inexpensive configuration and can perform a wide range of focus offset without sacrificing focusing accuracy. .

  In order to solve the above-described problems and achieve the object, a microscope according to the present invention includes an objective lens, a focusing member that changes a relative distance between the objective member and a holding member on which a sample to be observed is placed. Means, an illumination optical system that projects the first illumination light onto the sample, and the observation light that is emitted from the sample and projected through the objective lens when the first illumination light is projected. An observation optical system that is an infinity correction optical system, a light source that oscillates a second illumination light having a wavelength different from that of the first illumination light and the observation light, and the second illumination light as the objective lens A first reflecting surface that is a boundary surface between the holding member and the sample, and a second reflecting surface that is a surface opposite to the first reflecting surface of the holding member. And the second light that has passed through the objective lens again. A focus detection optical system that collects the illumination light, a focus detector that detects the second illumination light collected by the focus detection optical system, and the second that is detected by the focus detector. A focal position calculating means for calculating a relative distance in the optical axis direction between the first or second reflecting surface and the focal position of the objective lens in accordance with the state of the illumination light, and observing the sample in the optical axis direction Based on the calculation result of the input means for setting the position and the focus position calculation means, the focusing means is controlled so as to match the observation position set by the input means with the focus position of the objective lens. Control means, and the focus detection optical system makes the second illumination light incident on the objective lens in the form of condensed light with the second illumination light projected onto the sample. The focusing position of the objective lens Having a shifting means for shifting the objective lens than the focal position.

  In the microscope, the objective lens is an immersion objective lens, and a shift amount of the condensing position of the second illumination light with respect to a focal position of the objective lens is a range of an observation position that can be set by the input unit. Or less than 1/2.

  In the microscope, the objective lens is a dry objective lens, and a shift amount of the condensing position of the second illumination light with respect to a focal position of the objective lens is within a range of observation positions that can be set by the input means. It is an amount obtained by adding a distance of ½ or less and the thickness of the holding member.

  In the microscope, the shift unit includes a lens that focuses the second illumination light emitted from the light source, and a moving unit that changes the shift amount by moving the position of the lens along the optical axis. It is characterized by having.

  In the microscope, the shift unit includes a zoom optical system capable of switching a focal length of the second illumination light emitted from the light source, and a switching unit that changes the shift amount by switching the focal length. It is characterized by having.

  In the above microscope, the shift unit includes a relay optical system including a plurality of lenses, and an adjustment unit that changes the shift amount by adjusting an interval between the plurality of lenses.

  According to the present invention, the shift means for causing the second illumination light to enter the objective lens in the state of the condensed light and shifting the condensing position of the second illumination light to the objective lens side with respect to the focal position of the objective lens. Is provided in the focusing optical system, so that a microscope capable of performing a wide range of focus offsets without sacrificing the focusing accuracy can be realized without increasing the size of the light receiving sensor and with an inexpensive and simple configuration.

FIG. 1 is a partial cross-sectional view schematically showing a configuration of a microscope according to Embodiment 1 of the present invention. 2 is a partial cross-sectional view taken along the line AA of FIG. FIG. 3 is a schematic diagram showing the configuration in the vicinity of the revolver and in the sensor head shown in FIG. FIG. 4 is a schematic diagram for explaining the operation of the sensor head shown in FIG. FIG. 5 is a graph showing the characteristics of the gravity center position of the spot image. FIG. 6 is a schematic diagram showing spot images at the respective condensing positions shown in FIG. FIG. 7 is a diagram for explaining the operation of the microscope in the first modification. FIG. 8 is a schematic diagram illustrating a configuration example of the focusing lens in the second modification. FIG. 9 is a diagram for explaining the operation of the relay optical system shown in FIG. FIG. 10 is a schematic diagram illustrating a partial configuration of the microscope according to the second embodiment. FIG. 11 is a diagram for explaining the state of the spot image in the second embodiment.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings. Note that the present invention is not limited by these embodiments. Moreover, in description of each drawing, the same code | symbol is attached | subjected and shown to the same part. It should be noted that the drawings are schematic, and the dimensional relationships and ratios of each part are different from the actual ones. Also between the drawings, there are included portions having different dimensional relationships and ratios.

(Embodiment 1)
FIG. 1 is a partial cross-sectional view schematically showing a configuration of a microscope according to Embodiment 1 of the present invention. FIG. 2 is a partial cross-sectional view of the inside of the microscope main body taken along line AA in FIG. In FIG. 2, the description of the epi-fluorescent tube 130 and the excitation filter 131 shown in FIG. 1 is omitted.

  As shown in FIGS. 1 and 2, the microscope 1 according to the first embodiment of the present invention includes a microscope main body 10 and a control device 20 that controls the operation of the microscope main body 10.

  The microscope body 10 is so-called inverted that observes the reflected light that is irradiated on the sample SP from above the sample SP and transmitted through the sample SP or reflected on the sample SP from below the sample SP. A microscope of the mold. The microscope main body 10 includes a sample stage 101 on which a container (glass bottom dish) 100 on which a sample SP is placed, a housing 102 disposed below the sample stage 101, and transmitted illumination provided on the housing 102. A light source 104 for transmitted illumination attached to the column 103, a light source 105 for epi-illumination attached to the housing 102, an autofocus sensor head (hereinafter also simply referred to as a sensor head) 106 attached to the housing 102, And an image sensor 107 such as a CCD.

The light source 104 is a light source used when observing the sample SP through transmission, and generates illumination light in the visible region.
In the transmissive illumination column 103, a mirror 111 for bending the illumination light emitted from the light source 104 in the direction of the sample stage 101 is provided. Further, a condenser lens 112 for converging the illumination light bent by the mirror 111 is attached to the transmission illumination column 103.

  The sample stage 101 is movably provided on a plane (XY plane) orthogonal to the optical path (transmission illumination optical path) L1 of the illumination light bent by the mirror 111. In addition, the sample stage 101 is provided with a focusing motor 108 driven by a focusing motor driving unit 109 that operates under the control of the control device 20, and is electrically controlled via the control device 20. It can move in the vertical direction along the optical path L1. The casing 102 is also provided with a focusing handle 110 that is used when a manual focusing operation is performed. By operating the focusing handle 110, the sample stage 101 is manually moved up and down. It is also possible.

  The sample stage 101 is provided with an opening 101a through which illumination light can be transmitted. A glass bottom dish 100 containing a sample SP is placed so as to cover the opening 101a. In the first embodiment, as the member for holding the sample SP, the glass bottom dish 100 having a bottom surface made of transparent glass and provided with a partition wall on the side surface is used. Any member such as a slide glass or a well plate may be used as long as the bottom surface is transparent and the sample SP can be held.

  On the optical path L1 below the sample stage 101 (inside the casing 102), objective lenses 121 and 121 ′, a dichroic mirror 123, and a fluorescence provided so as to be inserted into and removed from the optical path L1. A filter cassette 124, an imaging lens 125, an optical path switching prism 126, and a mirror 127 are disposed. The objective lens 121 and the imaging lens 125 constitute an infinity correction optical system. Here, the infinity correction optical system generally means that the light beam that has passed through the objective lens from the sample does not form an image on the objective lens but enters the imaging lens as an infinite parallel light beam and forms an intermediate image by the imaging lens. It is an optical system.

  The revolver 122 is provided with a plurality of holes (not shown), and objective lenses 121 and 121 'are inserted into the holes. 1 to 3 show a state in which the two objective lenses 121 and 121 ′ are inserted into the revolver 122, the revolver 122 has more ( For example, it is possible to provide five objective lenses.

  The revolver 122 is provided with a revolver motor 141 that is driven by a revolver motor drive unit 142 that operates under the control device 20 to rotate the revolver 122. The user can insert desired objective lenses 121 and 121 ′ into the optical path L <b> 1 by operating the control device 20. The revolver 122 can also be rotated manually.

  Further, the revolver 122 is provided with a revo hole position detector 143 that detects which hole is inserted into the optical path L1. The control device 20 holds in advance information associating which magnification objective lens 121, 121 ′ is inserted into which hole, and based on this information and the output signal from the rebo hole position detection unit 143. The magnification of the objective lens 121 currently inserted in the optical path L1 can be recognized.

  The dichroic mirror 123 reflects a wavelength component (infrared region) of laser light used for autofocus described later, and transmits other wavelength components (visible region). Specifically, transmitted illumination light (observation light) transmitted through the sample SP, epi-illumination light irradiated to the sample SP, epi-illumination light (observation light) reflected by the sample SP, and fluorescence generated from the sample SP. Light (observation light) or the like passes through the dichroic mirror 123.

  When the sample SP is stained with a fluorescent dye, the fluorescent filter cassette 124 selectively selects a wavelength component (excitation light) that can excite the fluorescent dye out of the incident illumination light emitted from the epifluorescent floodlight tube 130. An excitation filter 131 that passes through the sample SP, a dichroic mirror 132 that selectively reflects the excitation light in the direction of the sample SP, and selectively transmits the fluorescent light generated by excitation of the fluorescent dye in the sample SP, and a dichroic mirror And an absorption filter 133 that absorbs unnecessary wavelength components from the fluorescent light transmitted through 132. In the fluorescent filter cassette 124, the excitation filter 131, the dichroic mirror 132, and the absorption filter 133 are prepared so that they can be exchanged by a switching mechanism such as a turret or the like, depending on the fluorescent dye used. used.

  The optical path switching prism 126 branches part of the observation light of the sample SP that has passed through the objective lens 121 and makes it incident on the image sensor 107 provided on the observation optical path L2. The imaging lens 125 images the observation light branched by the optical path switching prism 126 on the light receiving surface of the image sensor 107. Observation light received by the image sensor 107 is converted into an electrical signal by the image sensor 107 and output to the control device 20 as image data of the sample SP.

  The mirror 127 reflects the observation light transmitted through the optical path switching prism 126 and enters the eyepiece lens 129 via a plurality of relay lenses 128 provided on the observation optical path L3. The user can observe an enlarged image of the sample SP through the eyepiece lens 129.

  On the other hand, the light source 105 is a light source used for fluorescence observation of the sample SP, and is constituted by, for example, a mercury lamp that generates illumination light in the ultraviolet region. In addition, an epi-fluorescent fluorescent tube 130 that guides the light generated by the light source 105 in the direction of the transmitted illumination light path L1 is provided in the housing 102. The fluorescent filter cassette 124 can be inserted at a position where the incident-light fluorescent light projection tube 130 and the transmitted illumination light path L1 intersect.

  The control device 20 is configured by, for example, a personal computer or a workstation. The control device 20 is used during the execution of the control unit 21, the input unit 22 that receives inputs of various commands and information and inputs them to the control unit 21, various programs executed by the control unit 21, and the programs. A memory 23 for storing various information and a display unit 24 are provided.

  The control unit 21 is realized by hardware such as a CPU, for example, and reads and controls a predetermined program stored in the memory 23 to control the entire operation of the microscope 1.

  The input unit 22 may include input members such as various operation buttons in addition to an input device such as a keyboard and a mouse. For example, the input unit 22 rotates the revolver 122 to move the objective lens conversion switch for inserting the objective lenses 121 and 121 ′ having a desired magnification into the optical path L1, and the sample stage 101 is moved in the vertical direction along the optical path L1. And an auto focus switch for setting and canceling an auto focus operation. The input unit 22 receives signals input by operations on these input devices and input members, and outputs them to the control unit 21.

The memory 23 is realized by, for example, a ROM that stores a control program for the microscope 1 and a RAM that is a volatile memory that stores data necessary for the control as needed.
The display unit 24 is a display such as an LCD or an organic EL, and displays various images and information under the control of the control unit 21.

  In addition, the control device 20 includes an I / O port for inputting / outputting control signals and a data bus (none of which is shown) for connecting these components to each other. The control device 20 is connected to peripheral devices such as various motor drive units, light source drive units (oscillators), and address decoders (all described later) connected to the control device 20 via these I / O ports and data buses. Control.

  The control device 20 is provided with a jog encoder 26 and a pulse counter 27 as input means for changing the focus offset value. The encoder signal of the jog encoder 26 is converted into the number of pulses by the pulse counter 27 and input to the control unit 21. The control unit 21 reads the number of pulses from the pulse counter 27 to determine how much the jog encoder 26 is rotated in which direction, and changes the focus offset value according to the rotation amount of the jog encoder 26.

  The means for inputting the change of the focus offset value is not limited to the jog encoder 26. For example, a button that supports increase / decrease of the focus offset value may be provided on the input unit 22. In this case, the control unit 21 may set the change of the focus offset value according to the number of times the button is pressed, the pressing time, and the like.

  Next, the internal configuration and operation of the sensor head 106 will be described in detail. FIG. 3 is a schematic diagram showing a configuration in the vicinity of the revolver 122 and the sensor head 106 of the microscope body 10. FIG. 4 is a schematic diagram for explaining the operation of the sensor head 106.

  The sensor head 106 includes a reference light source 150 that oscillates laser light used for autofocus (hereinafter referred to as AF light), a light source driving unit 151 that drives the reference light source 150 under the control of the control unit 21, and a reference One of the collimated lenses 152 provided on the optical axis of the AF light emitted from the light source 150, the polarization beam splitter 153, the focusing lens 154, the λ / 4 wavelength plate 155, and the collimated lens 152. A projection-side stopper 156 that cuts a portion, a focusing lens 157 disposed on the optical path of the AF light reflected by the polarization beam splitter 153, a position detector (PSD) 158, an amplifier 159, And an A / D converter 160.

  As the reference light source 150, a light source capable of oscillating laser light in the visible light wavelength region such as infrared rays is used. The reference light source 150 performs pulse lighting under the control of the light source driving unit 151 and is controlled in strength.

The collimating lens 152 collimates the AF light emitted from the reference light source 150.
The light projecting side stopper 156 cuts half of the collimated AF light on a plane orthogonal to the optical axis of the AF light.

  The polarization beam splitter 153 transmits the P-polarized component of the remaining uncut AF light, guides it in the direction of the dichroic mirror 123, and reflects the S-polarized component of the incident light from the direction of the dichroic mirror 123. To the direction of the focusing lens 157.

The focusing lens 154 slightly focuses the AF light transmitted through the polarization beam splitter 153.
The λ / 4 wavelength plate 155 converts the transmitted AF light from linearly polarized light to circularly polarized light, or from circularly polarized light to linearly polarized light. In the sensor head 106, the λ / 4 wavelength plate 155 is arranged such that the optical axis is 45 ° with respect to the direction of linearly polarized light that has passed through the polarization beam splitter 153.

The focusing lens 157 collects the AF light reflected by the polarization beam splitter 143 and forms an image on the position detector 158.
The position detector 158 has a light receiving surface on which AF light is incident, and outputs a voltage signal corresponding to the position of the spot where the AF light is imaged.
The amplifier 159 amplifies the voltage signal output from the position detector 158 with a predetermined amplification factor.
The A / D converter 160 converts the amplified voltage signal into a digital value and outputs the digital value to the control device 20.

  The AF light emitted from the reference light source is collimated by the collimator lens 152, and then half of the light is cut by the light projection side stopper 156 (in FIG. 3, the upper half of the drawing). The remaining AF light passes through the polarization beam splitter 153 to become P-polarized light, is slightly focused by the converging lens 154, and then arranged so that the optical axis is 45 ° with respect to the direction of the linearly polarized light. It is converted into circularly polarized light by the / 4 wavelength plate 155. The AF light is reflected by the dichroic mirror 123 and condensed at one point by the objective lens 121 to form a spot-like image on the sample SP or in the vicinity thereof.

  At this time, in the observation optical system, the collimated light is condensed by the objective lens 121 and condensed on the sample SP, whereas the AF light is slightly focused by the focusing lens 154, so that the AF light is condensed. The position is shifted to the objective lens 121 side (the lower side of the drawing in FIG. 3) rather than the focal position of the objective lens 121 (condensing position of the observation light).

  Here, when observing a biological specimen such as a cell, since the sample SP to be observed generally has a three-dimensional structure, the user can enter the inside of the sample SP, that is, the focus reference plane (for example, the sample SP and the glass). A position offset from the sample SP side (upward in the drawing) along the optical path L1 from the boundary surface with the bottom dish 100) is observed. For this reason, in the first embodiment, in order to shift the focusing position of the AF light toward the objective lens 121 with respect to the focusing position of the observation light, the AF light slightly focused by the focusing lens 154 is used as the objective. The light is incident on the lens 121.

  The AF light reflected by the sample SP passes through the λ / 4 wavelength plate 155 via the objective lens 121 and the dichroic mirror 123, and at this time, the circularly polarized light is changed to S polarized light. The S-polarized AF light passes through the focusing lens 154, is reflected by the polarization beam splitter 153, is collected by the focusing lens 157, and forms an image on the light receiving surface of the position detector 158. Thereby, a voltage signal corresponding to the position of the spot of the AF light is output from the position detector 158. The voltage signal is amplified by the amplifier 159 at a predetermined amplification factor, converted to a digital value by the A / D converter 160, and output to the control device 20 as spot image plane data of AF light. Based on the spot image plane data, the control device 20 calculates the relative distance in the optical axis direction between the boundary surface C1 and the focal position of the objective lens 121, and executes autofocus.

  Next, a method for determining the shift amount δ of the focusing position of the AF light with respect to the focal position of the objective lens 121 will be described with reference to FIGS. In each of FIGS. 4A to 4C, the upper diagram shows an enlarged view of the vicinity of the focusing position of the observation light and the AF light, and the lower diagram shows the light receiving surface of the position detector 158 in the situation of the upper diagram. 2 shows a spot image of AF light that forms an image. In FIG. 4, the description of the sample stage 101 is omitted.

  In the first embodiment, a case will be described in which the sample SP in the glass bottom dish 100 is a biological specimen, and the objective lens 121 is an immersion system (for example, oil immersion type) objective lens. In this case, as shown in FIG. 4, the space between the glass bottom dish 100 and the objective lens 121 is filled with the immersion oil LQ.

  In general, the refractive index of a biological specimen is in the vicinity of the refractive index of water (1.3), and is substantially uniform inside the sample SP. The refractive index of the glass bottom dish 100 and the immersion oil LQ is about 1.5. Therefore, the reflectance at the boundary surface C1 between the glass bottom dish 100 and the sample SP is about 0.4%, and the reflectance at the boundary surface C2 between the immersion oil LQ and the glass bottom dish 100 is about 0%. For this reason, the AF light condensed on the sample SP or its vicinity by the objective lens 121 is hardly reflected at the boundary surface C2, but is mainly reflected at the boundary surface C1.

  Under such circumstances, as shown in FIG. 4A, it is assumed that the condensing position of the observation light and the illumination light (the focal position of the objective lens 121) coincides with the boundary surface C1. In this case, the focusing position of the AF light is below the boundary surface C1, that is, a point before the AF light reaches the boundary surface C1. For this reason, AF light is once condensed and then reflected on the boundary surface C1 in a diffused state. Accordingly, the range of the spot image IM1 of the AF light is widened to some extent, and the light intensity per unit area is weakened by the spread. The center of gravity position G of the spot image IM1 is a position deviated from the center C of the image plane of the position detector 158 (a point corresponding to the optical axis L4 of the AF light).

  Next, as shown in FIG. 4B, the objective lens 121 slightly approaches the sample SP, the condensing positions of the observation light and the illumination light enter the sample SP, and the condensing position of the AF light is the boundary surface C1. Is assumed to match. In this case, the spot image IM2 of the AF light is condensed at one point on the center C of the image plane, and the light intensity becomes the strongest.

  Further, as shown in FIG. 4C, the objective lens 121 is further brought closer to the sample SP, the condensing position of the observation light and the illumination light enters a deeper position of the sample SP, and the condensing position of the AF light is also a boundary surface. A case is assumed in which the sample SP is entered beyond C1. In this case, the AF light is reflected at the boundary surface C1 in a diffused state before being condensed. Accordingly, the range of the spot image IM3 of the AF light is expanded to some extent, and the light intensity per unit area is weakened by the extent of the spread.

  In this way, the light intensity per unit area and the gravity center position G of the spot image formed on the light receiving surface of the position detector 158 are changed by changing the condensing position of the observation light (illumination light) and AF light with respect to the sample SP. Changes.

FIG. 5 is a graph showing the characteristic of the gravity center position of the spot image with respect to the AF light condensing position. In FIG. 5, the horizontal axis indicates the focus position of the AF light on the optical path L1 (Z axis). The vertical axis represents the y coordinate of the barycentric position G of the spot image on the light receiving surface of the position detector 158. This barycentric position (y-coordinate value) is calculated from the luminance distribution on the light receiving surface based on the output signal of the position detector 158. Also, FIG. 6 (a) ~ (g) show spot images, respectively, in focusing position P _a to P _g shown in FIG.

As shown in FIG. 5, the characteristic of the center of gravity position of the spot image is that the condensing position P _{d where} y = 0 (the state where the AF condensing position coincides with the boundary surface C1: (see FIG. 4B) ) Appears symmetrically.

Among these, in FIGS. _{6B to} 6F corresponding to the condensing positions P _{b to} P _f , the spot image becomes larger as the condensing position is away from P _d , and the gravity center position G is separated from the center C. To go. Therefore, the area between the condensing position P _b to P _f in FIG. 5 has a linear region where the gravity center position G is changed linearly.

In FIGS. _6A , _6B , _6F , and _6G corresponding to the light condensing regions P _a , P _b , P _f , and P _g , the spot image partially protrudes from the light receiving surface. , It extends to half the area of the light receiving surface. For this reason, the gravity center position G is substantially the center position of the half area of the light receiving surface and does not change much. Therefore, the regions of the light collection positions P _{a to} P _b and P _{f to} P _g in FIG. 5 are plateau regions where the change in the center of gravity position G is small.

  Note that the outside of the plateau region is a region where effective output intensity (luminance information) is not detected because the light intensity per unit area of the spot image is lowered and buried in the dark noise of the electrical system.

Among such characteristics of the center of gravity position, in the linear region (condensing positions P _{b to} P _f ), the focus position of the AF light and the center of gravity position G are uniquely determined, so that focus offset can be performed. Become. However, in consideration of an error at the end of the linear region and taking a margin, it is actually preferable to set a part of the linear region (for example, a region of 1/2 or less) as the focus offset range. Therefore, in the first embodiment, it is set to a range of about half of the linear region (condensing position P _b to P _f) condensing position P _c to P _e as the focus offset range. That is, the configuration of the optical system including the focusing lens 154 is determined so that the shift amount δ of the focusing position of the AF light with respect to the focal position of the objective lens 121 becomes δ = | P _c −P _d |.

Next, the autofocus operation in the microscope 1 will be described.
When an autofocus switch provided in the input unit 22 is pressed, the control unit 21 outputs a control signal for starting the operation to the light source driving unit 151. In response to this, the light source driving unit 151 starts oscillation of the reference light source 150. The AF light emitted from the reference light source 150 is condensed on the sample SP or the vicinity thereof via the collimating lens 152, the polarizing beam splitter 153, the focusing lens 154, the λ / wavelength plate 155, the dichroic mirror 123, and the objective lens 121. .

  The control unit 21 also outputs a control signal that causes the focusing motor driving unit 109 to start operation. In response, the focusing motor driving unit 109 drives the focusing motor 108 to move the sample stage along the Z direction, and changes the relative distance of the objective lens 121 with respect to the sample stage 101.

Here, while the AF light condensing position is in the linear region (condensing positions P _{b to} P _f ) shown in FIG. 5, the relationship between the gravity center position G and the AF light condensing position is uniquely determined. In the meantime, the unit 21 takes in the output signal of the position detector 158 and calculates the gravity center position G of the spot image. Then, the calculated center-of-gravity position G is compared with an AF target value that is set for each magnification of the objective lens and is stored in advance in the memory 23, and the focusing motor drive unit using a known comparison control or the like from the difference. By controlling 109, autofocus is executed.

When the autofocus operation is performed from outside the range of the condensing positions P _{b to} P _f , the control unit 21 first operates the focusing motor drive unit 109 while monitoring the output signal of the position detector 158. A region where the light intensity is greater than or equal to a predetermined value is roughly searched. Then, when the AF light condensing position enters the linear region (condensing positions P _{b to} P _f ), the linear region may be searched in detail by the above-described comparison control or the like.

  Further, by continuously and repeatedly executing the chasing operation by the proportional control, it is possible to correct a focus drift that is normally generated due to a change in environmental temperature, heat generation of the microscope body, or the like as time passes. That is, since the focus position of the microscope can be stably maintained over time, for example, it is very effective for time-lapse observation in which changes in a living cell sample are observed over a long period of time.

Next, the focus offset set during the execution of the autofocus operation described above will be described.
During the autofocus operation, the control unit 21 monitors the jog encoder 26 based on the output signal of the pulse counter 27. When a signal indicating rotation is generated in the jog encoder 26, the control unit 21 changes the focus offset value according to the rotation amount. More specifically, the control unit 21 updates the AF target position by adding a focus offset value corresponding to the rotation amount to the AF target value set for each objective lens 121 and stored in advance in the memory 23. . Then, the current PSD output value is compared with the updated AF target value, and the focusing motor driving unit 109 is controlled by using comparison control or the like so that the difference becomes zero, whereby the sample stage 101 is detected. Move. By repeatedly executing such an operation, a focus offset value is additionally set with respect to the focus position detected by the autofocus operation, and the user can focus on a portion that the user wants to observe.

  As described above, when the focus offset value or the AF target position is changed based on the signal input from the jog encoder 26 by the user's operation, the control unit 21 sets the changed focus offset value or AF target position. Further, it may be stored in the memory 23 in association with the objective lens 121 inserted in the optical path L1 at that time. In this case, even after the objective lens 121 is replaced once, when the same objective lens 121 is used again, the autofocus operation is resumed with respect to the changed focus offset value based on the information stored in the memory 23. Can be made. Therefore, the user can start observing the sample SP from a state in which the focus is always accurately observed without resetting the focus offset value every time the objective lens 121 is replaced. In addition, since chromatic aberration that differs for each objective lens is also reflected in the AF target position, high-precision observation can always be performed.

Here, as described above, in the first embodiment, the execution range of the focus offset is limited. That is, the lower limit of the condensing position P _c is the focus offset, the condensing position P _c and symmetrical converging position P _e is the upper limit of the focus offset with respect to the condensing position P _d. Therefore, when the focus offset value changed based on the signal input from the jog encoder 26 by the user's operation reaches the upper limit or lower limit of the execution range, a warning lamp, a buzzer, etc. (not shown) This may be notified to the user.

  Next, the reason why the focus offset is performed by shifting the focal position of the objective lens 121 from the focusing position of the AF light to the objective lens 121 side in the first embodiment will be described.

In a general-purpose microscope using an area sensor or a line sensor for detecting AF light, the focal position of the objective lens and the focusing position of the AF light are almost the same even though there is chromatic aberration on the order of submicron. It is configured as follows. That is, in this case, since a shift amount δ is approximately 0 in the condensing position of the AF light to the focal position of the objective lens 121, when the focusing position of the AF light is in the Z = P _d shown in FIG. 5, an objective lens The focal position 121 is on the boundary surface C1 shown in FIG. However, where you want to observer observed, usually, since the upper side (the inside of the sample SP) than the boundary surfaces C1, space is available for focus offset is above the Z = P _d shown in FIG. 5 Limited. In other words, even though there is a linear region in which the relationship between the gravity center position G and the focusing position of the AF light is uniquely determined from Z = _Pb to Z = _Pf , a region that can be used effectively is Z = _Pd. To Z = P _f .

In the case of performing the focus offset in the microscope having such a configuration, in order to secure a wide range of focus offset amounts, it is necessary to enlarge the linear region (condensing positions P _{b to} P _f ) in the characteristics of the center of gravity shown in FIG. There is.

  One possible method for this is to use a larger sensor as a sensor for detecting AF light. As a result, as shown in FIG. 6, even when the spot image is enlarged away from the state where the focusing position of the AF light coincides with the boundary surface C1 (the state of FIG. 6D). The change of the spot image can be captured sufficiently.

  However, in this case, an increase in cost due to an increase in the size of the sensor is caused. Even if the sensor is enlarged, the light intensity per unit area of the spot image decreases in inverse proportion to the area. For this reason, when the spot image is gradually enlarged, even if the center of gravity position changes linearly at first, it is considered that the change of the center of gravity position is buried in noise before reaching the plateau region.

  In order to solve this problem, it is conceivable to change the intensity of the AF light according to the stage of the autofocus operation, or to improve the S / N of the sensor or amplifier, but the apparatus configuration becomes complicated. After all, it leads to cost increase.

  As another method for securing a wide range of focus offset amounts, it is conceivable to relax the slope of the linear region in the center of gravity position characteristic. This is realized by reducing the NA of AF light focused from the objective lens 121 toward the sample SP. However, in this case, the sensitivity of the sensor to changes in the focusing position of the AF light becomes dull. Therefore, there is a trade-off with the focusing accuracy of autofocus.

On the other hand, in the first embodiment, the focus offset range can be divided into a necessary amount by intentionally shifting the focusing position of the AF light to the objective lens 121 side rather than the focal position of the objective lens 121. Can only take widely. That is, the linear region in the characteristic of the center of gravity position is determined by the specifications of the position detector 158, but according to the first embodiment, it corresponds to the lower side than the boundary surface C1, which has not been effectively used in the past. the linear region (Z = P _b ~P _d in FIG. 5) will allow effective use.

  As described above, according to the first embodiment, a microscope capable of performing a wide range of focus offsets without taking measures such as increasing the size of a sensor for detecting AF light or improving S / N. In addition, it can be realized with an inexpensive configuration without sacrificing the focusing accuracy.

(Modification 1)
Next, a case where a dry objective lens is used in the microscope 1 having the same configuration as that of the first embodiment will be described with reference to FIG.
In this case, since the space between the glass bottom dish 100 and the objective lens 121 is filled with air (refractive index 1), the reflectance at the boundary surface C1 between the glass bottom dish 100 and the sample SP is about 0.4%. On the other hand, the reflectance at the boundary surface C2 between the glass bottom dish 100 and the air is overwhelmingly large. For this reason, AF light condensed on the sample SP or its vicinity by the objective lens 121 is almost reflected at the boundary surface C2.

  Under such circumstances, first, as shown in FIG. 7A, a case is assumed where the condensing position of the observation light and the illumination light (the focal position of the objective lens 121) coincides with the boundary surface C1. In this case, the focus position of the AF light is below the boundary surface C2, that is, a point before the AF light reaches the boundary surface C2. For this reason, the AF light is once condensed and then reflected again at the boundary surface C2 in a diffused state, so that a spot image IM1 ′ having a certain range is obtained, and the center of gravity position G is shifted from the center C. It becomes.

  Next, as shown in FIG. 7B, the objective lens 121 slightly approaches the sample SP, the condensing position of the observation light and the illumination light enters the sample SP, and the condensing position of the AF light is the boundary surface C2. Is assumed to match. In this case, the spot image IM2 'is condensed at one point of the center C of the image plane, and the light intensity becomes the strongest.

  Further, as shown in FIG. 7C, the objective lens 121 is further brought closer to the sample SP, the condensing position of the observation light and the illumination light enters a deeper position of the sample SP, and the condensing position of the AF light is also a boundary surface. A case is assumed in which the sample SP is entered beyond C1. In this case, since the AF light is reflected at the boundary surface C2 in a diffused state before being condensed, the range of the spot image IM3 'is expanded to some extent, and the center of gravity position G is shifted from the center C.

When such a dry objective lens is used, when the focus position of the AF light is on the boundary surface C2 (see FIG. 7B), the center of the linear region ( This is the condensing position P _d ). Therefore, in this case, the shift amount δ ′ of the focusing position of the AF light with respect to the focal position of the objective lens is set to a distance of about ½ of the focus offset range (condensing positions P _{b to} P _f ). What is necessary is just to set to the quantity which added thickness (DELTA) c of 100.

(Modification 2)
In the first embodiment and the first modification thereof, the shift amounts δ and δ ′ of the focusing position of the AF light with respect to the focal position of the objective lens are different between the immersion objective lens and the drying objective lens. Therefore, the focusing lens 154 is configured by a zoom optical system capable of switching the focal length manually or electrically, and switching for switching the shift amount between the focal length of the objective lens and the focusing position of the AF light in two or more steps. By providing the means, it is possible to realize a microscope that can be used for both the immersion objective lens and the dry objective lens. Alternatively, the shift amount between the focal length of the objective lens and the focusing position of the AF light may be changed by providing a moving unit that moves the focusing lens 154 along the optical axis.

(Modification 3)
The converging lens 154 shown in FIG. 3 may be configured by a relay optical system including two lenses as shown in FIGS. 8A and 8B, for example.
The configuration shown in FIG. 8A includes two positive lenses 171 and 172 disposed at positions close to the focal point, and a motor 173 that drives the lens 172 in the optical axis direction. On the other hand, FIG. 8B includes a positive lens 174 and a negative lens 175 arranged at positions close to the focal point, and a motor 176 that drives the lens 175 in the optical axis direction. In any configuration, the guide means for guiding the movement of the lenses 172 and 175 is omitted.

  According to such a relay optical system, the focusing state of AF light incident on the relay optical system is adjusted by moving one of the lenses constituting the relay optical system in the optical axis direction and adjusting the distance between the two lenses. And the focusing position of the AF light after passing through the objective lens 121 (see FIG. 3) can be changed to an arbitrary position. This action will be described with reference to FIG. 9 by taking as an example a relay optical system including positive lenses 171 and 172.

  For example, when the lens 172 is shifted by the length D in the left direction of the drawing as shown in FIG. 9B with respect to the arrangement of the lenses 171 and 172 shown in FIG. The focusing position of the AF light thus shifted is shifted by the length d in the right direction in the figure. The relationship between these shift amounts D and d is uniquely determined by the focal lengths and lens intervals of the lenses 171 and 172 and the objective lens 121. Therefore, according to such a relay optical system, the shift amount δ or δ ′ between the focal position of the objective lens 121 and the focusing position of the AF light can be set to an arbitrary amount.

  That is, when an immersion objective lens is used, the lens is arranged so that the focusing position of the AF light is shifted with respect to the focal position of the objective lens by a distance corresponding to about 1/2 of the focus offset possible range. The position 172 may be determined. When a dry objective lens is used, the distance of the objective lens is equal to the distance obtained by adding the thickness Δc of the glass bottom dish 100 to a distance corresponding to about ½ of the focus offset range. The position of the lens 172 may be determined so that the focusing position of the AF light is shifted with respect to the focal position. Accordingly, it is possible to realize a microscope that can be used with both the immersion objective lens and the dry objective lens.

  In the above description, the lenses 172 and 175 are electrically moved by the motors 173 and 176. However, a mechanism for manually moving the lenses 172 and 175 may be used. In the above description, the lenses 172 and 175 can be moved to arbitrary positions. However, a configuration in which the lenses 172 and 175 can be positioned stepwise in at least two types is possible. Furthermore, although the lens 172 side is moved in FIG. 8A, the lens 173 side may be moved. Similarly, although the lens 175 side is moved in FIG. 8B, the lens 174 side may be moved.

(Embodiment 2)
Next, a second embodiment of the present invention will be described.
FIG. 10 is a schematic diagram illustrating a partial configuration of the microscope according to the second embodiment.
As shown in FIG. 10, a microscope (hereinafter also simply referred to as a microscope) 1 according to the second embodiment is an autofocus sensor instead of the autofocus sensor head (hereinafter also simply referred to as a sensor head) 106 shown in FIG. A head 180 is provided. The configuration other than the autofocus sensor head 180 in the microscope according to the second embodiment is the same as that shown in FIGS.

  The sensor head 180 includes a collimator lens 181 instead of the collimator lens 152 and the light projection side stopper 156 shown in FIG. The reference light source 150 and the collimator lens 181 are arranged with the optical axes shifted from the polarization beam splitter 153 and the focusing lens 154, and AF light emitted from the reference light source 150 is collimated by the collimator lens 181. Then, the light passes through the polarization beam splitter 153 and enters the focusing lens 154.

  The sensor head 180 includes a CCD sensor 182 instead of the position detector 158 shown in FIG. 3 as a light receiving sensor for detecting a spot image of AF light. Other configurations of the sensor head 180 are the same as those in the first embodiment.

  In the case of such a configuration of the sensor head 180, as shown in FIG. 11, the spot images IM4 to IM6 formed on the light receiving surface of the CCD sensor 182 maintain a circular shape and the center of gravity according to the focus position of the AF light. Change position, deformation and light intensity. The CCD sensor 182 outputs an output signal representing the brightness of the detected spot image to the control unit 21.

  In this case, the control unit 21 calculates the gravity center position G of the spot images IM4 to IM6 based on the output signal from the CCD sensor 182. In the case of the CCD sensor 182, the barycentric position G of the spot images IM4 to IM6 can be output as a calculated value of pixels or less, and sufficient resolution can be obtained. Thereafter, the control unit 21 executes an autofocus operation and a focus offset operation using the gravity center position G in the same manner as in the first embodiment.

  As described above, according to the second embodiment, a wide range of focus offsets can be performed with a simple configuration without increasing the size of the light receiving sensor that detects the spot image and without sacrificing the focusing accuracy. It becomes possible.

  In the first and second embodiments described above, the light receiving sensor for detecting a spot image is not limited to a two-dimensional PSD or CCD. For example, a one-dimensional PSD, a CCD, or a two-divided photodetector is used in the moving direction of the spot image. A light receiving sensor may be configured by being arranged along the line.

  In Embodiments 1 and 2, the focusing lens 154 may be omitted. In this case, by shifting the collimating lenses 152 and 181 in the optical axis direction, the AF light emitted from the reference light source 150 can be made incident on the objective lens 121 as focused light.

  In the first and second embodiments, the sample stage 101 is moved to change the relative distance from the objective lens 121 to adjust the focus. On the contrary, the sample stage 101 is fixed and the objective is fixed. The revolver 122 that holds the lens 121 may be moved. Alternatively, both the sample stage 101 and the revolver 122 may be moved.

  Embodiment described above is only an example for implementing this invention, and this invention is not limited to these. Further, the present invention can form various inventions by appropriately combining a plurality of constituent elements disclosed in the respective embodiments and modifications. The present invention can be variously modified in accordance with specifications and the like, and various other embodiments are possible within the scope of the present invention.

DESCRIPTION OF SYMBOLS 1 Microscope 10 Microscope main body 20 Control apparatus 21 Control part 22 Input part 23 Memory 24 Display part 26 Jog encoder 27 Pulse counter 100 Glass bottom dish 101a Opening 101 Sample stage 102 Case 103 Transmitting illumination support | pillar 104 105 Light source 106 Autofocus sensor head 107 Image sensor 108 Focusing motor 109 Focusing motor drive unit 110 Focusing handle 111 Mirror 112 Condenser lens 121, 121 ′ Objective lens 122 Revolver 123 Dichroic mirror 124 Fluorescence filter cassette 125 Imaging lens 126 Optical path switching prism 127 Mirror 128 Relay lens 129 Eyepiece lens 130 Epi-illumination fluorescent tube 131 Excitation filter 132 Dichroic mirror 133 Absorption filter 141 Revolver motor 142 Revolver motor drive unit 143 Revo hole position detection unit 150 Reference light source 151 Light source drive unit 152 Collimator lens 153 Polarizing beam splitter 154, 157 Focusing lens 155 λ / 4 wavelength plate 156 Light emitting side stopper 158 Position detector 159 Amplifier 160 A / D converter 171, 172, 174, 175 Lens 173, 176 Motor 180 Autofocus sensor head 181 Collimator lens 182 CCD sensor

Claims (6)

An objective lens;
Focusing means for changing a relative distance between the holding member on which the sample to be observed is placed and the objective lens;
An illumination optical system that projects the first illumination light onto the sample;
An observation optical system that is an infinite correction optical system for observing the observation light that is emitted from the sample and projected through the objective lens when the first illumination light is projected;
A light source that oscillates second illumination light having a wavelength different from that of the first illumination light and the observation light;
The second illumination light is projected onto the sample through the objective lens, and the first reflecting surface that is a boundary surface between the holding member and the sample is opposite to the first reflecting surface of the holding member. A focus detection optical system that collects the second illumination light that is reflected by one of the second reflecting surfaces that are the side surfaces and passes through the objective lens again;
A focus detector for detecting the second illumination light collected by the focus detection optical system;
A focal position calculation that calculates a relative distance in the optical axis direction between the first or second reflecting surface and the focal position of the objective lens according to the state of the second illumination light detected by the focus detector. Means,
Input means for setting an observation position in the optical axis direction of the sample;
Control means for controlling the focusing means so as to match the observation position set by the input means and the focal position of the objective lens based on the calculation result of the focal position calculation means;
With
The focus detection optical system causes the second illumination light projected onto the sample to be incident on the objective lens in the state of the condensed light, thereby determining the condensing position of the second illumination light. A microscope characterized by having a shift means for shifting to the objective lens side relative to the focal position of the lens. The objective lens is an immersion objective lens,
The amount of shift of the condensing position of the second illumination light with respect to the focal position of the objective lens is less than or equal to ½ of the range of the observation position that can be set by the input means. Microscope. The objective lens is a dry objective lens,
The shift amount of the condensing position of the second illumination light with respect to the focal position of the objective lens is the sum of the distance of 1/2 or less of the range of the observation position that can be set by the input means and the thickness of the holding member. The microscope according to claim 1, wherein the microscope has a predetermined amount. The shifting means is
A lens that focuses the second illumination light emitted from the light source;
Moving means for changing the shift amount by moving the position of the lens along the optical axis;
The microscope according to any one of claims 1 to 3, wherein: The shifting means is
A zoom optical system capable of switching a focal length of the second illumination light emitted from the light source;
Switching means for changing the shift amount by switching the focal length;
The microscope according to any one of claims 1 to 3, wherein: The shifting means is
A relay optical system composed of a plurality of lenses;
Adjusting means for changing the shift amount by adjusting the interval between the plurality of lenses;
The microscope according to any one of claims 1 to 3, wherein:

Images