JP5053691B2 - Specimen scanner device and specimen position detection method using the device - Google Patents

Description

  The present invention relates to a specimen scanner device capable of detecting a predetermined position on a slide glass placed on a stage.

  In Patent Document 1, each image information obtained by photographing the entire area of the slide glass with the low-magnification objective lens is divided into the real field size of the high-magnification objective lens, and the presence or absence of the specimen image in the divided area is detected. There has been disclosed a microscopic image photographing apparatus in which only a divided region that has been found to exist is photographed with a high-magnification objective lens, whereby a wide-field and high-definition microscopic image can be efficiently photographed and the image recording capacity is reduced.

Patent Document 2 discloses a microscope image photographing apparatus capable of photographing a minimum necessary area and a high-definition microscope digital image that can be used particularly as a teaching material for pathological diagnosis. In Patent Document 2, the observation range currently observed is divided into small sections, and each small section is input at an observation magnification larger than the current observation magnification, whereby a high-resolution microscope image is formed. Yes.
JP 2004-101871 A Japanese Patent Laid-Open No. 2005-266718

  In Patent Document 1 and Patent Document 2, the entire surface of the slide glass is imaged once at a low magnification, and the screening position is determined and designated for the captured image. For example, in Patent Literature, automatic determination is made as to whether or not a sample exists from the luminance value of a captured image. In Patent Document 2, a user designates a target specimen from an image.

  Therefore, in Patent Document 1 and Patent Document 2, it is difficult to specify the screening position of the transparent specimen, and it takes time to image the entire slide glass once. As a result, the specimen is damaged due to phototoxicity, particularly in the fluorescent specimen.

  In view of the above-described problems, the present invention provides a method for automatically discriminating a specimen position (cover glass) without fading a transparent specimen and a fluorescent specimen in which fading is feared.

A specimen scanner used in a microscope according to the present invention, in which a specimen is placed on a slide glass, the slide glass covered with a specimen holding member for holding the specimen is placed on a stage, and the specimen is observed The apparatus includes a measurement light source for irradiating laser light for focusing, scanning control means for scanning the laser light relative to the slide glass, and the laser light scanned on the slide glass. A light intensity detecting means for detecting the light intensity of the reflected light; a light intensity value of the reflected light at a position on the slide glass obtained by the light intensity detecting means; and the reflected light at a position on the specimen holding member. A sample holding member recognizing means for comparing the light intensity values of the sample holding member and recognizing the position of the sample holding member on the slide glass based on the comparison result. That.

In the specimen scanner device, the scanning control means moves the stage on which the slide glass is placed in a direction perpendicular to the optical axis of the laser light.
In the specimen scanner device, the scanning control means scans the laser light in a one-dimensional direction or a two-dimensional direction relative to the slide glass placed on the stage .

  The sample holding member recognizing means extracts the light intensity of the reflected light that is larger than a predetermined threshold, and based on the position on the slide glass corresponding to the extracted light intensity, holds the sample on the slide glass. The position of the member is recognized.

The specimen scanner device further includes Z coordinate position detection means for detecting a position of the slide glass or the specimen holding member in the optical axis direction based on a detection result by the light intensity detection means. .

In the specimen scanner device, the Z coordinate position detecting means is an active autofocus mechanism.
The specimen scanner device is mounted on a microscope.

A specimen scanner used in a microscope according to the present invention, in which a specimen is placed on a slide glass, the slide glass covered with a specimen holding member for holding the specimen is placed on a stage, and the specimen is observed The specimen position detection method by the apparatus relatively scans the laser light for focusing on the slide glass, detects the light intensity of the reflected light of the laser light scanned on the slide glass, The light intensity of the reflected light at a position on the slide glass is compared with the light intensity of the reflected light at a position on the specimen holding member, and the position of the specimen holding member on the slide glass is compared based on the comparison result. It is characterized by recognizing.

  By using the present invention, the specimen position (cover glass position) can be observed even with a transparent specimen during screening. Further, the specimen position (cover glass position) can be detected without fading the fluorescent specimen.

A specimen scanner device according to the present invention is a microscope that places a specimen on a slide glass, places the slide glass covered with a specimen holding member that holds the specimen on a stage, and observes the specimen. It is used.

A specimen scanner device according to the present invention includes a scanning control means, a light intensity detection means, and a specimen holding member recognition means.
The scanning control means scans the slide glass with a predetermined laser beam relatively. For example, the scanning control unit may move the stage on which the slide glass is placed in a direction perpendicular to the optical axis of the laser beam (for example, in this embodiment, a stage control unit Equivalent to 5).

The light intensity detecting means detects the light intensity of the reflected light of the scanned laser light (for example, in the present embodiment, it corresponds to the optical sensor 36).
The specimen holding member recognizing means is based on the position on the slide glass scanned by the scanning control means and the light intensity of the reflected light corresponding to the position detected by the light intensity detecting means. The position of the specimen holding member on the slide glass is recognized (for example, this embodiment corresponds to the control unit 18).

By comprising in this way, the position of the cover glass on a slide glass is detectable.
Further, the scanning control means can scan the laser light relative to the slide glass placed on the stage in a one-dimensional direction or a two-dimensional direction.

By comprising in this way, the position of the cover glass arbitrarily arranged on the slide glass can be detected.
Further, the specimen holding member recognition means extracts the light intensity of the reflected light that is larger than a predetermined threshold, and based on the position on the slide glass corresponding to the extracted light intensity, the sample holding member recognition means The position of the specimen holding member can be recognized.

By configuring in this way, noise components can be removed, so that the cover glass on the slide glass 3a can be more easily identified.
The specimen scanner device may further include Z coordinate position detection means. The Z coordinate position detection means detects the position of the slide glass or the specimen holding member in the optical axis direction based on the detection result by the light intensity detection means. The Z coordinate position detection means may be, for example, an active autofocus mechanism (for example, in this embodiment, an infrared semiconductor laser 26, a laser control unit 27, and a condenser lens group that realize an active autofocus function). 35 corresponds to the light receiving sensor 36 or the like).

  With this configuration, AF can be performed. Therefore, since the rate of change between the light intensity of the reflected light from the cover glass and the light intensity of the reflected light from the slide glass becomes larger, it becomes easier to determine whether the cover glass or the slide glass is used.

  FIG. 1 shows a configuration example of a microscope system to which a specimen scanner device according to the present invention is applied. In FIG. 1, in an inverted microscope system 1 as an example of a microscope system according to the present invention, a specimen 2 as a test object is placed on a slide glass 3a and covered with a cover glass 3b.

  Further, the slide glass 3 a on which the specimen 2 is placed is fixed to the electric stage 4. The electric stage 4 can be electrically controlled in the XY direction orthogonal to the optical axis, and the control is performed by the stage controller 5.

  The fluorescent light source 6 is a light source for performing fluorescent illumination on the specimen 2. The excitation light emitted from the fluorescent light source 6 is collected by the collector lens 7 and irradiated to the specimen 2 fixed on the electric stage 4 through the excitation filter 8, the dichroic mirror 9, and the objective lens 10a. The specimen 2 is illuminated by

  The fluorescence emitted from the specimen 2 by being illuminated with the excitation light passes through the dichroic mirror 9 and the absorption filter 11, and is guided to the eyepiece 13 or the CCD camera unit 14 by the optical path switching unit 12.

  The image of the specimen 2 captured by the CCD camera unit 14 is acquired by the host PC 16 by the video capture board 15. The host PC 16 stores the acquired image on an image memory (not shown). This image memory is usually capable of storing a plurality of images.

The electric shutter 17 is a shutter for shielding the excitation light emitted from the fluorescent light source 6, and the control unit 18 can control the shielding of the excitation light.
The objective lens 10 (10a, 10b) is attached to the electric revolver 19. The electric revolver 19 has a function of inserting an arbitrary objective lens 10a or objective lens 10b into the optical path by a signal from the control unit 18.

  The electric revolver 19 is fixed to a pedestal 23 driven by a focusing motor 22 via a piezo element 21 and is moved in the optical axis direction (Z direction) by a motor control unit 24 controlled from the control unit 18. By doing so, it is possible to relatively drive the distance between the sample 2 and the objective lens 10a.

  On the other hand, the piezo element 21 is disposed between the electric revolver 19 and the gantry 23, and the thickness of the piezo element 21 in the optical axis direction is electrically changed by a piezo drive unit 25 controlled by the control unit 18. The structure is such that the position of the objective lens 10a attached to the interlocking revolver 19 is moved in the direction of the optical axis. Similarly, the distance between the specimen 2 and the objective lens 10a can be relatively driven. The piezo element 21 is an element having characteristics of high speed and high resolution, although the driving range is shorter than the focusing motor 22.

  The control unit 18 is connected to the host PC 16 and performs various controls of the inverted microscope system 1. The control unit 18 is connected to a jog encoder 38 for a user to input an instruction for driving the electric revolver 19, a pulse counter 39, and an operation unit 40 for inputting other various instructions.

  On the other hand, the infrared light semiconductor laser 26 which is an invisible wavelength region corresponds to a measurement light source for performing focusing. The infrared semiconductor laser 26 is controlled by a laser control unit 27 connected to the control unit 18.

  Laser light emitted from the semiconductor laser 26 passes through a collimating lens 28 for maintaining parallel light, and half of the beam diameter is cut by the light projecting side stopper 29. Thereafter, only the P-polarized light component is reflected by a PBS (Polarization Beam Splitter) 30 and guided to the sample 2 side.

  The light beam once condensed by the condenser lens group 31 passes through the chromatic aberration correction lens group 32. The light that has passed through the chromatic aberration correction lens group 32 is polarized by 45 ° by the λ / 4 plate 33 and enters the dichroic mirror 34. Here, since only the infrared region is reflected by the dichroic mirror 34, the reflected light beam forms a spot-shaped image on the surface of the slide glass 3a or the cover glass 3b by the objective lens 10a.

  The light beam reflected by the slide glass 3a or the cover glass 3b is then further polarized by 45 ° by the λ / 4 plate 33 via the objective lens 10a and the dichroic mirror 34 and switched to the S-polarized component.

  Further, the light returns from the chromatic aberration correction lens group 32 and the condenser lens group 31 and enters the PBS 30. Then, since the light flux is an S-polarized component, it passes through the PBS 30 as it is, and passes through the condenser lens group 35 and is imaged on the light receiving sensor 36.

  The light receiving sensor 36 is a two-divided photodiode (A region and B region) arranged around the optical axis. When the sample 2 is in the focus position, as shown in FIG. 2B, the spot imaged on the light receiving sensor 36 is a narrow and high-intensity signal, and the sample 2 is in the Z direction from the focus position. When it is on the upper side (rear pin position), as shown in FIG. 2C, the signal intensity distribution is biased toward the range of the B region, and the sample 2 is lower than the focus position in the Z direction (front pin position). ), The signal intensity distribution is biased toward the range of the A region as shown in FIG. 2A, and each is converted into a sensor signal.

  Then, the detection signal divided and converted into the range of the A region and the B region is output to the focus determination unit 37. The focus determination unit 37 calculates the sum of the intensity in each range based on the input detection signal. That is, as shown in FIG. 3A, when the horizontal axis is the Z direction of the electric revolver 19 and the vertical axis is the light intensity incident on each light receiving sensor 36, the A range signal symmetrical to the left and right across the focus position and Two curves of the B range signal can be detected.

  Next, A + B as shown in FIG. 3B and (A−B) / (A + B) as shown in FIG. 3C are calculated from the A range signal and B range signal. In particular, the characteristic as shown in FIG. 3C is called an S-shaped curve, and its value is called an evaluation function value (hereinafter abbreviated as Ef value).

  The control unit 18 determines the focus position direction based on the sign of the Ef value from the focus determination unit 37. For example, when AF (autofocus) is started from the position “1” in FIG. 3C, the control unit 18 performs control to raise the electric revolver 19 because the sign of the Ef value is negative. For example, when AF is started from the position “2” in FIG. 3C, the control unit 18 performs control to lower the electric revolver 19 because the sign of the Ef value is positive. The control unit 18 performs such control, and the motor control unit 24 relatively drives the distance between the objective lens 10a and the surface of the slide glass 3 in the optical axis direction so that the Ef value finally becomes zero. To perform the focusing operation. In addition, the thing in the case of having two front and rear reflecting surfaces like the slide glass 3 also has a function of automatically focusing on the surface of the slide glass 3 from the relationship between the two coordinate positions.

As described above, the active light AF optical system is realized by controlling the lighting of the laser light and detecting the reflected light of the laser beam projected onto the observation sample.
FIG. 4 shows an example of the internal configuration of the control unit 18. A ROM (Read Only Memory) 18b stores a program for controlling the system. A RAM (Random Access Memory) 18c stores data necessary for control and is a volatile memory or the like.

The CPU (Central Processing Unit) 18a reads a preset program stored in the ROM 18b and executes various controls. The input / output (I / O) port 18d is an input / output interface for exchanging data with various drive units, various control units, determination units, and the like of the microscope 1. The external interface (I / F) 18e is a communication interface for enabling communication with an external device such as the operation unit 40 or a PC.

  FIG. 5 is a flowchart showing an outline of detecting the position of the cover glass 3b on the slide glass 3a in the present invention. FIG. 6 shows the slide glass 3 a placed on the electric stage 4. The flow of FIG. 5 will be described below with reference to FIG.

  First, the specimen 2 is placed on the slide glass 3a, and the top of the specimen 2 is covered with a cover glass 3b to create a slide glass 3a for microscope observation. Then, as shown in FIG. 6 (a), the slide glass 3a thus created is placed on the electric stage 4 and pressed by the Clenmel 4a to fix the slide glass 3a to the electric stage 4 (Step 1. Hereinafter, the steps are described as follows. Referred to as "S").

  Next, the infrared semiconductor laser 26 is scanned in a fixed direction from a predetermined position of the slide glass 3a by moving the electric stage 4 in a one-dimensional direction or a two-dimensional direction (S2). For example, as shown in FIG. 6B, the Z position of the electric stage 4 (for example, the position where the light intensity is maximized (the position where AF is applied)) and the Y position are fixed at predetermined positions. In the state, the infrared semiconductor laser 26 is scanned with respect to the X direction of the slide glass 3a. The active AF light intensity value at the time of scanning is held in the RAM 18c together with the X coordinate from which the light intensity value was detected. The magnification of the objective lens is fixed.

At this time, as will be described later, when detecting the position of the cover glass 3b, the light intensity of the slide glass is acquired in advance as the reference value _Pstd in order to identify whether the first scanned position is the slide glass or the cover glass. Keep it. As an acquisition method, for example, a specified position on the slide glass may be scanned, or the user may arbitrarily specify a predetermined position on the slide glass.

  Next, data of light intensity values larger than a predetermined threshold is extracted from the light intensity values acquired in S2 (S3). Thereby, the position of the cover glass 3b on the slide glass 3a can be recognized. This is because, as shown in FIG. 6B, the portion without the cover glass 3b (light intensity value P1) and the portion with the cover glass 3b (light intensity value P2) are reflected by the difference in refractive index. This is because the light intensity values of the reflected light are different. This will be described in detail with reference to FIG.

  FIG. 7 is a diagram for explaining the principle of the present invention. FIG. 7A shows that the laser beam irradiated from the infrared semiconductor laser 26 is irradiated from the objective lens 10 to the slide glass 3a by moving the electric stage 4 on which the slide glass 3a is placed in the X direction. The state of scanning in the X direction of the slide glass 3a is shown. The angle θ indicates the tilt angle of the slide glass. The angle θ is arbitrary, and may be 0 ° (not inclined) or may be inclined.

  FIG. 7B shows light intensity values detected at points a, b, c, d, e, and f at the sampling points on the scanning line scanned in FIG. 7A. . In FIG. 7B, a1 to f1 indicate the calculation results of A + B at points a to f in FIG. 7A, respectively (A + B has been described in FIG. 3B). a2 to f2 indicate the calculation results of (A−B) / (A + B) at points a to c, respectively ((A−B) / (A + B) has been described in FIG. 3C).

  In FIG. 7, the case where the slide glass 3a is inclined at an angle θ with respect to the mounting surface of the electric stage 4 will be described as an example. In this case, a straight line in which the Z position corresponding to the surface of the slide glass 3a inclined by the angle θ is plotted is represented by a broken line Z1, and a straight line in which the Z position corresponding to the surface of the cover glass 3b inclined by the angle θ is plotted. This is represented by a broken line Z2. W indicates the width of the cover glass 3b in the scanning direction.

In a1 and b1, a peak P1 exists at a predetermined Z position. Also in a2 and b2, the evaluation function values Ef corresponding to the Z position where the peak value P1 exists can be confirmed as α and β.
At c1 and d1, the peak P1 at the Z position focused on at a1 and b1 decreases to the peak P2, and a new peak is generated at a Z position different from the focused Z position. In C2, the peak value P2 after the decrease and the evaluation function value Ef corresponding to the Z position where the newly generated peak value exists can be confirmed as γ and δ, respectively. The evaluation function value Ef of d1 is omitted because it is substantially the same as c2.

  At e1 and f1, the peaks generated at c1 and d1 disappear, the peak P2 at the Z position focused on at a1 and b1 increases, and is restored to P1 again. The evaluation function values Ef of e1 and f1 are omitted because they are substantially the same as a2 and b2.

  In this way, the laser beam is scanned in a certain direction with respect to the slide glass 3a on which the cover glass 3b is set, and A + B and / or (A−B) / (A + B regarding the slide glass 3a part and the cover glass 3b part. ) Can be detected to detect a difference in light intensity and / or a change point. Therefore, the position of the cover glass 3b on the slide glass 3a can be detected by detecting the position on the slide glass 3a corresponding to the detected light intensity difference and / or change point.

  Thus, the position of the slide glass 3a on the slide glass 3a can be detected by utilizing the fact that the focus position changes due to the difference in refractive index between the slide glass 3a and the cover glass 3b. The details of the embodiment of the present invention will now be described.

<First Embodiment>
This embodiment demonstrates the case where a laser beam is scanned to a one-dimensional direction relatively with respect to a slide glass.

FIG. 8 is a top view of the slide glass for explaining the scanning direction in the present embodiment. In the figure, n cover glasses 3 (3b-1, 3b-2, ... 3b-n) are set on a slide glass 3a. The Y coordinate of an arbitrary position in the width in the transverse direction of the slide glass 3a (Y-direction) expressed in Y _c.

End of the Y-coordinate = Y _c on each coverslip 3b in the slide glass. 3a, XS1 for _{3b-1 (X 11, Y} c), expressed in _{XS2 (X 12, Y c)} , 3b-2 Table in _{XS1 (X 21, Y c)} , XS2 (X 22, Y c) is represented by, ..., for the _{3b-n XS1 (X n1,} Yc), XS2 (X n2, Y c) for Is done.

  FIG. 9 shows a flow for recognizing the position of the cover glass on the slide glass in the present embodiment. First, the user places the desired specimen 2 on the slide glass 3a and covers the specimen 2 with the cover glass 3b. This is repeated and a plurality of cover glasses 3b (3b-1, 3b-2,... 3b-n) are placed on the slide glass 3a as shown in FIG.

  Then, the user places the slide glass 3a on the electric stage 4 and presses it with the clemmel 4a (S11). In the present embodiment, it is assumed that the slide glass 3a has an inclination angle θ = 0 ° with respect to the electric stage 4.

Next, when the user presses a “cover glass position recognition” button (not shown) on the operation unit 40, a cover glass position recognition instruction signal is transmitted. When the control unit 18 receives the cover glass position recognition instruction signal, the control unit 18 scans the laser beam in the X direction at the _Yc position on the slide glass 3a (S12). The control unit 18 holds a predetermined Y position ( _Yc position) on the slide glass 3a in advance. In the present embodiment, the Z position and the Y position of the electric stage are fixed during scanning.

Specifically, the CPU 18 a reads the Y position (Y _c ) stored in the ROM 18 _b and transmits it to the stage control unit 5. The stage control unit 5 moves the electric stage 4 so that the tip (optical axis) of the objective lens 10 is positioned on the perpendicular line of Yc. The magnification of the objective lens is fixed.

At this time, the X coordinate corresponding to Y _c is arbitrary, but is usually set in advance so as to be near the end of the slide glass 3a (for example, the X coordinate corresponding to Y _c is stored in the ROM 18b). May be). In the present embodiment, as shown in FIG. 8, the coordinates (X ₀ , Y _c ) on the slide glass are set as the scan start position Q.

  Under the control of the stage controller 5, the scan start position Q of the slide glass 3 a on the electric stage 4 reaches the optical axis of the laser light emitted from the tip of the objective lens 10. Then, the control unit 18 drives the laser driving unit 27 to irradiate the infrared semiconductor laser 26, and the control unit 18 drives the stage driving unit 5 to move the electric stage 4 in the X direction.

  The control unit 18 stores the value PX (X) of the AF peak position (peak value of light intensity) for each X coordinate X (X) in the RAM 18c in accordance with the scanning in the X direction in S12 (S13).

  Specifically, the control unit 18 takes in the laser reflected light intensity for each X coordinate X (X) detected by the light receiving sensor 36 via the focus determination unit 37, calculates A + B as described above, The peak value PX (X) of the light intensity value is acquired.

  The sampling frequency may be variable. For example, the peak value PX (x) may be held for all the coordinates in the X direction on the slide glass 3a, or the peak value PX (X) may be held by skipping the coordinates. May be.

  In this embodiment, since the difference (change rate) between the light intensity of the reflected light from the cover glass 3b and the light intensity of the reflected light from the slide glass 3a is detected, it is not always necessary to perform AF. However, since the rate of change between the light intensity of the reflected light from the cover glass 3b and the light intensity of the reflected light from the slide glass 3a is increased by performing AF, it is possible to discriminate between the cover glass 3b and the slide glass 3a. It becomes easier.

  Next, the control part 18 compares each PX (X) value acquired by S13, and the position coordinate in which the cover glass 3b exists (a cover glass start point position is represented by XS1, and a cover glass end point position is represented by XS2). Is recognized (S14). The process of S14 will be described in detail with reference to FIG.

  FIG. 10 shows a graph of the light intensity value with respect to the scan direction when there is no cover glass at the scan start position Q in the present embodiment. FIG. 10 is a conceptual graph of the AF peak position (peak value of light intensity) PX (X) for each X coordinate X (X) held in S13 of FIG.

FIG. 10A is a top view of a slide glass on which a cover glass is placed.
FIG. 10B shows a graph of the light intensity value with respect to the scanning direction when the cover glass is focused. Since the cover glass is focused, the light intensity of the XS1-XS2 section with the cover glass is higher than that of the other sections.

  FIG. 10C shows a graph of light intensity values with respect to the scanning direction when the slide glass is focused. Since the slide glass is focused, the section XS1-XS2 where the slide glass surface is exposed and the section after XS2 (that is, the section not covered with the cover glass) are lighter than the other sections. Strength is getting stronger.

P _std indicates the light intensity at a specified position on the slide glass acquired in advance. “WS” is a threshold value for removing a noise component in the scanning direction. For example, a minimum width used as a cover glass is set. “WP _t ” is a threshold value for the AF peak position, and is a threshold value for removing noise components. “C” is a counter for measuring the cover glass width. Further, the value of P (1) + WP _t is represented by WP.

From the scan start position Q to XS1, the light intensity value P _std (P _std includes P _std + σ (σ: error range); the same applies hereinafter). From XS1 to XS2, in the case of FIG. 10B, the light intensity value P (N) <P _std −WP _t , and in the case of FIG. 10C, P (N)> P _std + WP _t ( N is a variable indicating the position on the slide glass, as will be described later.) Later XS2 is again light intensity value P _std.

  FIG. 11 shows a flow for recognizing the position and number of the cover glasses 3b on the slide glass 3a based on the scan result in the present embodiment (when the slide glass is previously focused). This flow is a detailed flow of S14 of FIG. This flow will be described using the variables described in FIG.

  First, variables are initialized (S21). For example, a counter variable (slide glass coordinate counter) N for measuring coordinates on the slide glass is set to N = 1, and a counter variable (cover glass width counter) C for measuring the width of the cover glass is set to C = 1. And the number of recognized cover glasses (cover glass recognition number) n is n = 0.

Next, it is determined whether or not the light intensity P _{std at} the specified position on the slide glass 3a acquired in advance is _equal to or greater than the light intensity P (N = 1) at N = 1 (S22). When the cover glass already exists at the scan start position Q, the light intensity P (1) decreases because the surface of the slide glass 3a is not focused, so that P (1) becomes P _std or more. There is no. Therefore, if the cover glass 3b already exists at the scan start position Q, the process proceeds to “No”. On the other hand, if there is no cover glass at the scan start position Q and the surface of the slide glass 3a is exposed, P (1) does not decrease, and the process proceeds to “Yes”.

  When N = 1 and the slide glass 3a is exposed (when “Yes” is reached in S22), it is determined whether or not the light intensity value WP <P (N−1) −P (N). (S23). The case of WP <P (N-1) -P (N) is a case where the scan position moves from the slide glass 3a onto the cover glass 3b. The case of WP ≦ P (N−1) −P (N) is a case where the slide glass surface is scanned. However, if it is the first time to perform the process of S23, that is, if N = 1, the process proceeds to “No” in S23. In this example, since N = 1, the process proceeds to “No” in S23.

  Next, the cover glass width counter C is reset to “1” (S35). After the process of S35, it is determined whether or not the slide glass coordinate counter N is smaller than the width of the slide glass 3a in the scanning direction (S33). In the present embodiment, the width of the slide glass 3a is set to 750, but the present invention is not limited to this.

  Since N <“750” (proceed to “Yes” in S33), N is incremented (S34). S23 → S35 → S33 → S34 → S23 →... Is repeated until WP <P (N−1) −P (N) is established.

  When WP <P (N−1) −P (N) is established (the portion where the cover glass 3b is present) (proceed to “Yes” in S23), it is determined whether or not the cover glass width counter C is “1”. (S24). If it is the first process for the cover glass 3b, since C = 1, the process proceeds to S25. If it is the second and subsequent processes, the process proceeds to S28 because C> 1.

  When the cover glass width counter C is “1” (proceed to “Yes” in S24), it is determined whether or not the cover glass recognition number n is “0” (S25). In the slide glass 3a, if the cover glass 3b is first recognized, n = 0, so the process proceeds to S26, but if not n> 0, the process proceeds to S27. When n = 0 (proceed to “Yes” in S25), n = 1 is set (S26), and the process proceeds to S27.

Next, the slide glass coordinate counter N is substituted into the cover glass starting point position XS1 (n) (S27).
Next, it is determined whether or not the cover glass width counter C is larger than the threshold value WS (S28). When cover glass width counter C ≦ threshold WS (proceed to “No” in S28), it is determined whether cover glass width counter C is equal to threshold WS (S29).

  If cover glass width counter C <threshold WS (proceed to “No” in S29), cover glass width counter C is incremented (S32), and since N <“750” (proceed to “Yes” in S33), N Increment (S34). Thereafter, S23 to S34 are repeated until the cover glass width counter C = threshold WS (S29) is established.

  When the cover glass width counter C = the threshold WS is established (goes to “Yes” in S29), the cover glass recognition number n is incremented (S30), and the slide glass coordinate counter N is set to the cover glass end point position XS2 (n−1). (S31).

Thereafter, the cover glass width counter C is incremented (S32), and since N <“750” (proceed to “Yes” in S33), N is incremented (S34).
In the process of S23, while WP <P (N-1) -P (N) (that is, while the cover glass is being processed), C continues to be incremented (S32), and the cover glass end point position XS2 ( n-1) is continuously updated (S31).

  Then, when there is no cover glass, WP ≧ P (N−1) −P (N) (the process proceeds to “No” in S23), so the cover glass width counter C is reset to “1” (S35), S23 to S35 are repeated during N <“750”.

  When N = 750, that is, when the slide glass coordinate counter N becomes equal to the width of the slide glass 3a in the scan direction (proceed to "No" in S33), this flow ends.

  In this way, the number of cover glasses 3b (cover glass recognition number n) present on the slide glass 3a and the positions (XS1, XS2) of the recognized cover glasses can be detected.

Next, detection of a cover glass when it is already a cover glass at the scan start position Q will be described.
FIG. 12 shows a graph of the light intensity value with respect to the scan direction when the cover glass already exists at the scan start position Q in the present embodiment. FIG. 12 corresponds to the case where the cover glass start position XS1 of FIG.

FIG. 12A is a top view of the slide glass on which the cover glass is placed at the scan start position.
FIG. 12B shows a graph of the light intensity value with respect to the scanning direction when the cover glass is focused. Since the focus is on the cover glass, the Q (= XS1) -XS2 section with the cover glass has a higher light intensity than the other sections.

  FIG. 12C shows a graph of light intensity values with respect to the scanning direction when the slide glass is focused. Since the slide glass is in focus, the Q (= XS1) -XS2 section where the slide glass surface is exposed and the section after XS2 (that is, the section not covered with the cover glass) are in other sections. Compared with the light intensity.

Since the scan glass is scanned from the scan start position Q (= XS1) to XS2, in the case of FIG. 12B, the light intensity value P (N) <P _std -WP _t , FIG. ), P (N)> P _std + WP _t (N is a variable indicating the position on the slide glass, as will be described later). Later XS2 is a light intensity value P _std.

  In the flow of FIG. 11, if the cover glass 3b already exists at the scan start position, the process proceeds to “No” in S22. Thereafter, as described above, S23 to S34 are performed until the cover glass disappears, and when the cover glass disappears, WP ≧ P (N−1) −P (N) (“No” in S23). ), The cover glass width counter C is reset to “1” (S35), and S23 to S35 are repeated while N <“750”.

  When N = 750, that is, when the slide glass coordinate counter N becomes equal to the width of the slide glass 3a in the scan direction (proceed to "No" in S33), this flow ends.

  Thus, even when the cover glass is already at the scan start position Q, the number of cover glasses 3b existing on the slide glass 3a (cover glass recognition number n) and the positions of the recognized cover glasses (XS1) , XS2) can be detected.

  According to the present embodiment, when screening a specimen (cover glass) on a slide glass, only one line or a plurality of lines are scanned in a predetermined direction instead of the entire slide glass, so that only the specimen is efficiently screened in a short time. can do. By the screening operation, the position of the cover glass on the slide glass with the specimen can be detected in a short time. Further, by detecting the position of the cover glass, the position of the specimen on the slide glass can be roughly understood, so that efficient microscopic observation can be performed.

  In addition, since the imaging process is not performed when the cover glass is detected, there is no need to irradiate the specimen with illumination light, and therefore there is no phototoxicity to the specimen. Further, since it is not necessary to use image processing when recognizing the position of the specimen, it can be applied to a transparent specimen.

  In the present embodiment, the case where the slide glass is focused in advance has been described with reference to FIG. 11, but the present embodiment can be similarly applied to the case where the cover glass is focused in advance.

<Second Embodiment>
This embodiment demonstrates the case where a laser beam is scanned in a two-dimensional direction relatively with respect to a slide glass. In this embodiment, after the cover glass position in the X direction is recognized by performing the processing of the first embodiment, each recognized cover glass is scanned in the Y direction to recognize the position in the Y direction. To do.

  FIG. 13 is a top view of the slide glass for explaining the scanning direction in the present embodiment. As described in FIG. 8, since the X coordinate (cover glass start point position XS1, cover glass end point position XS2) of each cover glass is obtained according to the first embodiment, the center coordinates XSC of XS1 and XS2 are calculated, Laser scanning is performed in the Y direction from the center coordinate XSC.

  FIG. 14 shows a flow for recognizing the position of the cover glass on the slide glass in the present embodiment. Similar to the first embodiment, S11 to S14 are executed to recognize the cover glass position in the X direction.

  Next, the control unit 18 calculates the center coordinate XSC of the coordinate position XS1-XS2 with the cover glass for each cover glass recognized in S14, and executes scanning from the center coordinate XSC in the Y direction (S41). . S41 is the same as S12 except that the scanning direction is different.

  Next, with the scan in the Y direction in S41, the control unit 18 holds the value PY (Y) of the AF peak position (light intensity peak value) for each Y coordinate: Y (Y) in the RAM 18c ( S42). S42 is the same as S13 except that the scanning direction is different.

  Next, the control unit 18 compares the respective PY (Y) values acquired in S42, and the position coordinates of the cover glass 3b in the Y direction (the cover glass start point position is represented by YS1, and the cover glass end point position is represented by YS2). .) Is recognized (S43). S43 is the same as S14 (and FIG. 11) except that the scanning direction is different.

  FIG. 15 shows an example of a modification of the first and second embodiments. In the first embodiment, the Z position and the Y position are fixed and scanning is performed once in the X direction. However, as shown in FIG. 15A, the Y position may be changed and scanning may be performed a plurality of times in the X direction. . Further, as shown in FIG. 15B, the position coordinates (XS1-XS2) of the cover glasses may be scanned a plurality of times in the Y direction.

Further, the shape of the cover glass is not limited to a square, and may be a rectangle or a polygon, may be a circle as shown in FIG. 15C, or may be an ellipse.
Further, the cover glass on the slide glass is not limited to the one-dimensionally arranged one, but may be arranged two-dimensionally as shown in FIG. 15 (d), or arranged radially or concentrically. It may be.

  In this way, when screening a specimen (cover glass) on a slide glass, only one line or a plurality of lines are scanned in a predetermined direction (one-dimensional direction or two-dimensional direction) instead of the entire slide glass. Screening can be performed automatically and efficiently in a short time.

  According to this embodiment, in addition to the effects of the first embodiment, since scanning in a two-dimensional direction can be performed, even if the cover glass is arranged two-dimensionally on the slide glass, the slide The specimen position on the glass (the position of the cover glass) can be automatically detected to save labor in the screening work.

  Although the embodiments of the present invention have been described above, the present invention is not limited to the above-described embodiments, and various improvements and modifications can be made without departing from the scope of the present invention. .

  In the embodiment of the present invention, the sample scanner device is described as an example integrated with a microscope, but is not limited thereto. For example, the specimen scanner device of the present embodiment may include a microscope and a separate body, for example, a scanning control means, a light intensity detection means, and a specimen holding member recognition means.

  As described above, according to the present invention, even when a transparent specimen is used, the specimen position (cover glass position) can be specified and observed during screening. Further, the specimen position (cover glass position) can be detected without fading the fluorescent specimen.

1 shows a configuration example of a microscope system to which a sample scanner device according to the present invention is applied. It is a figure for demonstrating the relationship between a light receiving sensor and a focus position. It is a figure for demonstrating the relationship between defocus and incident light intensity. An example of an internal structure of the control part 18 is shown. It is a flow which shows the outline | summary which detects the position of the cover glass 3b on the slide glass 3a in this invention. The slide glass 3a mounted on the electric stage 4 is shown. It is a figure for demonstrating the principle of this invention. It is a top view of the slide glass for demonstrating the scanning direction in 1st Embodiment. The flow which recognizes the position of the cover glass on the slide glass in 1st Embodiment is shown. The graph of the light intensity value with respect to the scanning direction when there is no cover glass at the scan start position Q in the first embodiment is shown. The flow which recognizes the position and the number of the cover glasses 3b on the slide glass 3a based on the scan result in the first embodiment is shown. The graph of the light intensity value with respect to the scanning direction when there is already a cover glass at the scan start position Q in the present embodiment is shown. It is a top view of the slide glass for demonstrating the scanning direction in 2nd Embodiment. The flow which recognizes the position of the cover glass on the slide glass in 2nd Embodiment is shown. An example of the modification of 1st and 2nd embodiment is shown.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Inverted microscope system 2 Sample 3a Slide glass 3b Cover glass 4 Electric stage 5 Stage control part 6 Fluorescence light source 7 Collector lens 8 Excitation filter 9 Dichroic mirror 10 (10a, 10b) Objective lens 11 Absorption filter 12 Optical path switching part 13 Eyepiece 14 CCD camera unit 15 Video capture board 16 Host PC
DESCRIPTION OF SYMBOLS 17 Electric shutter 18 Control part 19 Electric revolver 21 Piezo element 22 Semi-focusing part motor 23 Base 24 Motor control part 25 Piezo control part 26 Infrared semiconductor laser 27 Laser control part 28 Collimating lens 29 Light emission side stopper 30 PBS
Reference Signs List 31 Condensing Lens Group 32 Chromatic Aberration Correction Lens Group 33 λ / 4 Plate 34 Dichroic Mirror 35 Condensing Lens Group 36 Light Receiving Sensor 37 Focus Determination Unit 38 Jog Encoder 39 Pulse Counter 40 Operation Unit

Claims (8)

In a specimen scanner device used in a microscope for placing a specimen on a slide glass, placing the slide glass covering the specimen with a specimen holding member that holds the specimen on a stage, and observing the specimen,
A measurement light source that emits a laser beam for focusing;
Scanning control means for scanning the laser beam relative to the slide glass;
A light intensity detecting means for detecting the light intensity of the reflected light of the laser beam scanned on the slide glass;
The light intensity value of the reflected light at the position on the slide glass obtained by the light intensity detection means is compared with the light intensity value of the reflected light at the position on the specimen holding member, and based on the comparison result, A specimen holding member recognition means for recognizing the position of the specimen holding member on the slide glass;
A specimen scanner device comprising: The specimen scanner device according to claim 1, wherein the scanning control unit moves a stage on which the slide glass is placed in a direction perpendicular to the optical axis of the laser light. 2. The specimen scanner device according to claim 1, wherein the scanning control unit scans the laser light relatively in a one-dimensional direction or a two-dimensional direction with respect to the slide glass placed on the stage. . The sample holding member recognizing means extracts the light intensity of the reflected light that is larger than a predetermined threshold, and based on the position on the slide glass corresponding to the extracted light intensity, holds the sample on the slide glass. The specimen scanner device according to claim 1, wherein the position of the member is recognized. The specimen scanner device further includes:
The specimen scanner according to claim 1, further comprising a Z-coordinate position detecting means for detecting a position of the slide glass or the specimen holding member in the optical axis direction based on a detection result by the light intensity detecting means. apparatus. The specimen scanner device according to claim 5 , wherein the Z coordinate position detection means is an active autofocus mechanism. A microscope comprising the specimen scanner device according to any one of claims 1 to 6 . Place a specimen on a slide glass, place the slide glass covered with a specimen holding member holding the specimen on the stage, and detect the specimen position by a specimen scanner device used in a microscope for observing the specimen In the method
The laser beam for focusing on the slide glass is relatively scanned,
Detecting the light intensity of the reflected light of the laser beam scanned on the slide glass,
The light intensity of the reflected light at a position on the slide glass is compared with the light intensity of the reflected light at a position on the specimen holding member, and the position of the specimen holding member on the slide glass is compared based on the comparison result. Recognize
A specimen position detection method characterized by the above.