JP2006030304A - Focus detector for microscope - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a focus detector for a microscope accurately detecting a focusing position even when a peak shape obtained when relation between the intensity of a light quantity signal and a relative position is made a graph, is flat. <P>SOLUTION: Based on a data row showing the relation between the position of an objective and the intensity of the light quantity signal, a position Z<SB>0</SB>corresponding to the maximum intensity MAX of the light quantity signal is obtained and is set as a reference position. Positions Z<SB>1</SB>and Z<SB>-1</SB>separate from the reference position by a 1st distance D<SB>1</SB>and positions Z<SB>2</SB>and Z<SB>-2</SB>separate from the reference position by a 2nd distance D<SB>2</SB>different from the 1st distance are set. Then, the intersection (k) of a line La linking two points corresponding to the position Z<SB>1</SB>and the position Z<SB>2</SB>and a line Lb linking two points corresponding to the position Z<SB>-1</SB>and the position Z<SB>-2</SB>out of respective points in the data row is obtained, and the position at the intersection is decided as the focusing position Z<SB>f</SB>. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a focus detection apparatus for a microscope that performs focus detection by capturing weak light generated from an observation object, and is particularly suitable for performing focus detection by capturing weak fluorescence generated from an object for fluorescence observation. The present invention relates to a focus detection apparatus for a microscope.

  In a fluorescence microscope, an autofocus mechanism may be used to form a clear fluorescent image of an observation object on a predetermined imaging surface (for example, an imaging surface of an image sensor). A focus detection device is incorporated in the autofocus mechanism, and a position shift (focus shift) between a fluorescent image formed by an observation optical system of a microscope and a predetermined image plane is detected. This detection result is used as an index when adjusting the relative position between the objective lens of the observation optical system and the observation object in the autofocus mechanism.

The focus detection apparatus of the fluorescence microscope includes a sensor unit and a calculation unit, and converts weak fluorescence generated from the observation object into an electrical signal (hereinafter referred to as “light amount signal”). Then, while changing the relative position between the objective lens and the observation object, the light amount signal from the sensor unit is taken into the calculation unit, and the relative position corresponding to the maximum intensity of the light amount signal is determined as the in-focus position (for example, patent) Reference 1).
JP 2004-4634 A

  However, the conventional focus detection apparatus described above is based on the premise that a clear peak shape appears when the relationship between the intensity of the light amount signal and the relative position is graphed. For this reason, the intensity of the background component of the light amount signal increases due to the influence of fluorescence generated from the periphery of the observation object (for example, a solvent or a culture solution), and when the above peak shape becomes relatively flat, It was difficult to specify the maximum intensity, and the focus position could not be detected accurately.

  An object of the present invention is to provide a focus detection device for a microscope that can accurately detect a focus position even when the peak shape when the relationship between the intensity of a light amount signal and the relative position is graphed is flat.

  The microscope focus detection apparatus according to claim 1, wherein a sensor unit that detects focus detection light generated from an observation object and outputs a light amount signal corresponding to a relative position between the object and the objective lens; A light generation signal that captures the light amount signals at a plurality of the relative positions from the sensor unit and generates a data string representing a relationship between the relative position and the intensity of the light amount signal, and the light amount signal based on the data string A first setting unit that obtains the relative position corresponding to the maximum intensity and sets the relative position as a reference position; and is spaced apart from the reference position by a first interval in a direction in which the relative position approaches the reference position. A first position and a second position separated from the reference position by a second interval different from the first interval are set, and in the direction in which the relative position is separated from the reference position, A second setting unit for setting a third position separated by the first interval and a fourth position separated by the second interval from the reference position; and the first position among the points of the data string And the straight line connecting the two points corresponding to the second position and the straight line connecting the third point and the two points corresponding to the fourth position are determined, and the relative position at the intersection is determined as the in-focus position And a determination unit.

  According to a second aspect of the present invention, in the focus detection apparatus for a microscope according to the first aspect, the microscope further includes a determination unit that determines a peak shape when the data string is graphed, and the first setting unit and the first setting unit When the peak shape is flat as a result of the determination by the determination unit, the second setting unit and the determination unit, respectively, the reference position setting process, the first position, the second position, and the A setting process for the third position and the fourth position and a determination process for the in-focus position are executed.

According to a third aspect of the present invention, in the focus detection apparatus for a microscope according to the second aspect, the determination unit determines that the maximum intensity MAX of the light amount signal and the intensity MIN of the background component of the data string are as follows: When the conditional expression is satisfied, the peak shape is determined to be flat.
MAX / MIN <1.05
According to a fourth aspect of the present invention, in the microscope focus detection apparatus according to any one of the first to third aspects, the generation unit outputs the light amount signals at the plurality of relative positions from the sensor unit. The data string is generated by performing a moving average process of the intensities of the plurality of light quantity signals that are captured and the relative positions are adjacent to each other.

  According to a fifth aspect of the present invention, in the microscope focus detection apparatus according to the fourth aspect, the generation unit determines the number of executions of the moving average process based on a sensitivity setting value of the sensor unit. is there.

  According to the focus detection apparatus for a microscope of the present invention, it is possible to accurately detect the in-focus position even when the peak shape is flat when the relationship between the intensity of the light amount signal and the relative position is graphed.

Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.
The focus detection apparatus 10 (FIG. 1) of the present embodiment is a focus detection apparatus of a fluorescence microscope, and performs focus detection by capturing weak fluorescence generated from an object (specimen 21) for fluorescence observation. The specimen 21 is, for example, a biological specimen (DNA, protein, etc.) labeled with a fluorescent substance.
As shown in FIG. 1, the focus detection apparatus 10 according to the present embodiment includes a light amount detection circuit 11 and a drive circuit 12 that are arranged inside a casing 20 of a fluorescence microscope, and a storage circuit 13 that is arranged outside the casing 20. , An in-focus position calculation circuit 14 and a position control circuit 15. The light quantity detection circuit 11 is provided with a photomultiplier tube (not shown).

  In addition, a fixed stage 22 that supports the specimen 21, a movable objective lens 23, a moving mechanism 24 that moves the sample up and down, a light source 25, a dichroic mirror 26, and a half mirror 27 are provided inside the housing 20. Further, a second objective lens and a wavelength selection filter (not shown) are provided. Similar to the dichroic mirror 26 and the half mirror 27, the second objective lens and the wavelength selection filter are disposed on the optical axis 23a of the objective lens 23.

  Incidentally, the optical characteristics of the dichroic mirror 26 are such that the illumination light from the light source 25 (that is, the excitation light for the fluorescent material in the specimen 21) is selectively reflected and the fluorescence generated from the fluorescent material is selectively transmitted. It is a characteristic. The optical characteristics of the wavelength selective filter are such that the excitation light is selectively blocked and the fluorescence is selectively transmitted. The wavelength selection filter is disposed between the dichroic mirror 26 and the half mirror 27, for example.

  The focus detection apparatus 10 of the present embodiment has a positional deviation between the fluorescence image of the specimen 21 formed by the observation optical system of the fluorescence microscope (that is, the objective lens 23 and the second objective lens (not shown)) and the predetermined imaging plane 28. This is a device for detecting (defocus). This detection result is used as an index when adjusting the position of the objective lens 23 (that is, the relative position between the objective lens 23 and the specimen 21). If the objective lens 23 is in the in-focus position, a clear fluorescent image of the specimen 21 can be formed on the predetermined image plane 28. For example, an imaging surface of an image sensor is disposed on the imaging surface 28.

  At the time of focus detection, illumination light from the light source 25 (excitation light for the fluorescent substance in the specimen 21) is irradiated to the specimen 21 on the stage 22 via the dichroic mirror 26 and the objective lens 23. At this time, it is preferable that the irradiation direction of the illumination light is an oblique direction with respect to the optical axis 23 a of the objective lens 23. For such oblique illumination, for example, a technique disclosed in Japanese Patent Application Laid-Open No. 2004-4634 can be employed. In addition to the oblique illumination, the specimen 21 may be irradiated with illumination light parallel to the optical axis 23a.

  The focus detection fluorescence generated from the specimen 21 by the illumination is incident on the photomultiplier tube of the light amount detection circuit 11 through the objective lens 23, the dichroic mirror 26, the half mirror 27, and a wavelength selection filter (not shown). To do. The photomultiplier tube is a sensor unit suitable for detecting weak light, and includes a condensing lens for efficiently guiding the reflected light from the half mirror 27 to the light receiving surface. When the oblique illumination described above is employed, the light receiving surface of the photomultiplier tube is disposed behind a surface conjugate with a predetermined imaging surface 28.

  The photomultiplier tube of the light amount detection circuit 11 collectively detects the focus detection fluorescence incident on the light receiving surface and outputs a voltage signal (hereinafter referred to as “light amount signal”) proportional to the light amount to the storage circuit 13 at the subsequent stage. To do. The storage circuit 13 temporarily stores the intensity (voltage value) of the light amount signal output from the light amount detection circuit 11. Such a series of processing by the light amount detection circuit 11 and the storage circuit 13 (that is, storage processing of the intensity of the light amount signal) is synchronized with the movement processing of the objective lens 23 by the position control circuit 15 and the drive circuit 12 described below. Repeatedly.

  The position control circuit 15 is a numerical value related to the position of the objective lens 23 in order to change the position of the objective lens 23 (that is, the relative position between the objective lens 23 and the sample 21) in the vertical direction along the optical axis 23a during focus detection. Information is generated and a control signal based on the numerical information is output to the drive circuit 12 at the subsequent stage. The drive circuit 12 generates a drive signal for the moving mechanism 24 based on the control signal from the position control circuit 15. Then, the moving mechanism 24 is driven using this drive signal, and the position of the objective lens 23 (that is, the relative position between the objective lens 23 and the specimen 21) is changed along the optical axis 23a.

  In the focus detection apparatus 10 of the present embodiment, the photomultiplier tube of the light amount detection circuit 11 is changed at each of a plurality of different positions while changing the position of the objective lens 23 (that is, the relative position between the objective lens 23 and the specimen 21). The intensity (voltage value) of the light amount signal output from is stored in the storage circuit 13. When such a scanning operation is completed, calculation processing (described later) based on the intensity of a large number of light quantity signals is performed by the in-focus position calculation circuit 14. The storage circuit 13 and the in-focus position calculation circuit 14 generally correspond to a “generation unit” in the claims. The in-focus position calculation circuit 14 corresponds to “first setting unit”, “second setting unit”, and “determination unit” in the claims.

Next, the detection operation of the focus detection apparatus 10 of this embodiment will be described using the flowchart of FIG.
In step S1, pre-scanning is performed, and the movement range of the objective lens 23 (the range of the main scan in the next step S2) is determined so as to include the in-focus position of the objective lens 23. In the present embodiment, the case where the intensity of the light amount signal output from the photomultiplier tube of the light amount detection circuit 11 is equal to the intensity of the background component at the upper limit end and the lower limit end of the movement range of the objective lens 23 will be described. The background component is not a fluorescence generated from the sample 21 but a component corresponding to the fluorescence generated from the periphery of the sample 21 (for example, a solvent or a culture solution). In the present embodiment, the distance between the upper limit end and the lower limit end of the movement range of the objective lens 23 is at least 240 μm (for example, about 300 μm).

  In step S2, the main scan is performed according to the range determined in step S1. In other words, the photomultiplier of the light quantity detection circuit 11 at each of a plurality of different positions while changing the position of the objective lens 23 from the lower limit end to the upper limit end (or from the upper limit end to the lower limit end) of the movement range of the objective lens 23. The intensity (voltage value) of the light amount signal output from the tube is stored in the storage circuit 13. At this time, it is preferable to capture a light amount signal from the photomultiplier tube every time the position changes by a certain interval within the movement range of the objective lens 23.

  When the position of the objective lens 23 reaches the end point, and when the capturing of the light amount signal at the end point is finished, the storage circuit 13 temporarily stores a plurality of light amount signals with different positions of the objective lens 23. . The intensity (voltage value) of each light quantity signal has a different magnitude depending on the position of the objective lens 23. Each of these light quantity signals is output to the subsequent focus position calculation circuit 14 in order to determine the focus position of the objective lens 23.

  In step S <b> 3, when the in-focus position calculation circuit 14 takes in a plurality of light amount signals with different positions of the objective lens 23 from the storage circuit 13, it generates a data string representing the relationship between the position of the objective lens 23 and the intensity of the light amount signal. Then, this data string (raw data) is graphed. That is, a matrix graph is created. An example of the matrix graph is shown in FIG. In FIG. 3A, the horizontal axis represents the position of the objective lens 23, and the vertical axis represents the intensity (V) of the light amount signal. One convex portion appears in the matrix graph.

  Next (step S4), moving average processing of the data string (raw data) generated in step S3 is performed. That is, the moving average value Vave of the intensity is calculated using a plurality of light quantity signals adjacent to the position of the objective lens 23 at each point in the data string. Then, the moving average value Vave is replaced with the current value. If the number of light quantity signals used for calculating the moving average value Vave is f, the moving average value Vave can be calculated by the following equations (1) and (2).

Vave = _{[V (n-f 0) +} ... + V (n) + ... + V (n + f 0) ] / f ... (1)
f ₀ = (f−1) / 2 (2)
As a result of such moving average processing, the high-frequency component of the data string (raw data) is removed, and a data string with a good SN ratio is newly generated. Further, the matrix graph of FIG. 3A is smoothed and becomes, for example, a thick line of FIG.

  Next (step S5), the SN determination of the data string after the moving average process is performed. For this determination, for example, a set value of the applied voltage (that is, sensitivity) of the photomultiplier tube of the light amount detection circuit 11 may be used. If the sensitivity of the photomultiplier tube is low, the influence of noise is small, and if the sensitivity is high, it is considered that the influence of noise is likely to occur. Therefore, the SN determination of the data string can be performed according to the sensitivity setting value.

  And as a result of SN determination, when further noise removal is necessary (S5 is No), the process returns to step S4 to perform the second moving average process. The processing method is the same as the first time. As a result of the second moving average process, the high-frequency component of the data string (after the first moving average process) is removed, and a data string with a better SN ratio is newly generated. The matrix graph in FIG. 3B is further smoothed, for example, as shown by the thick line in FIG.

  The number of executions of the moving average process in steps S4 and S5 may be determined according to the set value of the sensitivity of the photomultiplier tube. After the required number of times (one or more times) of moving average processing is performed, if the SN determination is OK (Yes in S5), the in-focus position calculation circuit 14 generates the last generated data string (one or more times of moving average) The process after the next step S6 is performed using (after process). Note that the last generated data string (simply referred to as “data string” in the following description) has a good SN ratio from which high-frequency components have been sufficiently removed, and corresponds to the “data string” in the claims.

  In the peak shape when the data string is graphed (for example, the peak shape of the matrix graph shown in FIGS. 3B and 3C), the intensity of the light amount signal at the both ends of the movement range of the objective lens 23 (that is, the minimum intensity MIN). Is equal to the intensity of the background component (the component corresponding to the fluorescence generated from the periphery of the specimen 21 (for example, solvent or culture medium)). Further, the difference between the minimum intensity MIN and the maximum intensity MAX of the light amount signal in the data string is a component corresponding to the fluorescence generated from the specimen 21.

  In step S6, in order to determine the level (flatness) of the peak shape (for example, the peak shape of the matrix graph shown in FIGS. 3B and 3C) when the data row is graphed, the light amount signal of the data row is determined. A ratio (MAX / MIN) between the maximum intensity MAX and the minimum intensity MIN is calculated. When the peak ratio MAX / MIN satisfies the following conditional expression (3) (Yes in S6), it is determined that the peak shape is low (flat), and the process proceeds to step S7 (four-point expression calculation). . Conversely, when the peak ratio MAX / MIN does not satisfy the conditional expression (3), it is determined that the peak shape is high, and the process proceeds to step S8 (normal calculation).

MAX / MIN <1.05 (3)
The process of step S7 (4-point calculation) will be described. The data string in this case has a low peak shape when graphed, and the peak ratio MAX / MIN is less than 1.05. The focus position calculation circuit 14 obtains the position Z ₀ of the objective lens 23 corresponding to the maximum intensity MAX of the light quantity signal based on such a data string (FIG. 4), and sets this position Z ₀ as a reference position. . The reference position Z ₀ is a reference for four-point calculation and is not necessarily the in-focus position of the objective lens 23.

Then, the reference position Z ₀ from front focus side (direction where the objective lens 23 approaches the sample 21), the reference position and Z ₀ position Z ₁ separated by intervals D ₁ from the reference position Z ₀ from spacing D ₂ (> A position Z ₂ separated by D ₁ ) is set. Further, the rear focus side from the reference position Z ₀ (direction objective lens 23 is away the specimen 21), and a position Z _-1 from the reference position Z ₀ apart intervals D _1, from the reference position Z ₀ by a distance D ₂ away Set the position Z- ₂ .

In the present embodiment, for example, the interval D ₁ is 60 μm and the interval D ₂ is 120 μm. In this case, the reference position Z ₀ and the four positions Z ₋₂ , Z ₋₁ , Z ₁ , Z ₂ are set at intervals of 60 μm. Distance between the position Z _-2 across the position Z ₂ is 240 .mu.m. Note that the movement range (the range of the main scan) of the objective lens 23 determined in step S1 is equal to or greater than the interval (240 μm) between the position Z ₋₂ and the position Z ₂ .

Four positions Z _-2, Z _-1, when Z _1, Z ₂ of the setting is completed, the focusing position calculating circuit 14, of each point of data row, point 2 corresponding to the position Z ₁ to the position Z ₂ E ₁ and E ₂ are selected, and a straight line La connecting the two points E ₁ and E ₂ is obtained. Further, two points E ₋₁ and E ₋₂ corresponding to the position Z ₋₁ and the position Z ₋₂ are selected, and a straight line Lb connecting the two points E ₋₁ and E ₋₂ is obtained. Then, an intersection point K between the straight line La and the straight line Lb is obtained, and the position of the objective lens 23 corresponding to the intersection point K is defined as “focus position (Z _f )”.

The specific calculation is performed according to the following equation (4). In equation (4), information about four points E ₁ , E ₂ , E ₋₁ , E ₋₂ selected from the data string (that is, positions Z ₁ , Z ₂ , Z ₋₁ , Z ₋₂ and each position) The in-focus position (Z _f ) can be obtained by substituting the corresponding light intensity signal intensities P ₁ , P ₂ , P ₋₁ , P ₋₂ ).

次に、ステップＳ８の処理（通常計算）について説明する。この場合のデータ列は、グラフ化したときのピーク形状が高く、ピーク比率ＭＡＸ／ＭＩＮが１.０５以上となっている。合焦位置演算回路１４は、このようなデータ列（図５）に基づいて、光量信号の最大強度ＭＡＸより小さい強度Ｑ（＝(１−ｎ％)×ＭＡＸ）に対応する２点Ｅ_Q1,Ｅ_Q2を選択し、この２点Ｅ_Q1,Ｅ_Q2の中点Ｅ_QCを求め、中点Ｅ_QCに対応する対物レンズ２３の位置を“合焦位置（Ｚ_f）”とする。ｎ＝２〜４％程度が好ましい。なお、ステップＳ８の処理（通常計算）は、２点Ｅ_Q1,Ｅ_Q2のみを計算に用いるので、上記ステップＳ７の処理（４点式計算）と比較して短時間で計算を行える。

Next, the process (normal calculation) in step S8 will be described. The data string in this case has a high peak shape when graphed, and the peak ratio MAX / MIN is 1.05 or more. Based on such a data string (FIG. 5), the in-focus position calculation circuit 14 has two points E _Q1 , corresponding to an intensity Q (= (1−n%) × MAX) smaller than the maximum intensity MAX of the light amount signal. E _Q2 is selected, the midpoint E _QC of these two points E _Q1 and E _Q2 is obtained, and the position of the objective lens 23 corresponding to the mid point E _QC is set as the “focus position (Z _f )”. n = 2 to 4% is preferable. In addition, since only the two points E _Q1 and E _Q2 are used for the calculation in step S8 (normal calculation), the calculation can be performed in a shorter time compared to the process in step S7 (four-point calculation).

In this way, when the peak shape is low (flat), the process of step S7 (4-point calculation) is performed, and when the peak shape is high, the process of step S8 (normal calculation) is performed, When the calculation of the in-focus position (Z _f ) has been completed, the in-focus position calculation circuit 14 determines whether or not the calculation result is appropriate in the next step S9. This determination corresponds to determination of whether or not the calculation result (that is, the in-focus position (Z _f )) is within the movement range (main scan range) of the objective lens 23.

If an appropriate calculation result can be obtained (Yes in S9), the calculation result is determined as “focus position (Z _f )” in the next step S10. If the calculation result is not appropriate (S9 is No), the process proceeds to step S11 to retry. Retry is required when the peak shape is very small (when the matrix graph is flat), when the sample 21 does not exist by mistake, or when the peak shape includes two convex portions. One of the two convex portions is attributed to fluorescence from the specimen 21, while the other is considered to be attributed to fluorescence from the periphery of the specimen 21 (for example, a solvent or a culture solution).

In step S11, it is determined whether or not this retry is the first time. Only in the case of the first retry (S11 is Yes), the process returns to step S2 (main scan) again through step S12. In step S12, the setting of the moving speed of the objective lens 23 in the second main scan is changed to a low speed.
For this reason, in step S2, the light quantity signal can be taken from the photomultiplier tube of the light quantity detection circuit 11 at a sampling interval finer than the first time within the same range (movement range of the objective lens 23) as the first time. As a result, in the next step S3, a data string (raw data) with higher accuracy than the first time can be generated. Thereafter, the same steps S4 to S6 and S7 (or S8) as described above are repeated to calculate the in-focus position (Z _f ) based on a highly accurate data string after one or more moving average processes. be able to.

In the next step S9, it is determined whether or not the calculation result (that is, the in-focus position (Z _f )) by such a retry is appropriate. If it is appropriate (S9 is Yes), the calculation result is “matched”. The focal position ( _Zf ) "is determined (S10). Usually, when the peak shape is very small (when the matrix graph is flat), the focus position (Z _f ) can be determined at this stage.
However, if the sample 21 is not present by mistake or if the peak shape includes two convex portions, an appropriate calculation result cannot be obtained even if the first retry is performed (No in S9). In this case, the process proceeds to step S11, and since it is the second retry (S11 is No), an undetectable error process (step S13) is performed.

As described above, in the focus detection apparatus 10 of the present embodiment, the peak shape (for example, the matrix shown in FIGS. 3B and 3C) when the relationship between the intensity of the light amount signal and the position of the objective lens 23 is graphed. Since the processing of step S7 (four-point calculation) is performed when the peak shape of the graph is low, the in-focus position (Z _f ) can be accurately detected even when the peak shape is low. Further, according to the processing of step S7 (four-point calculation), the straight lines La and Lb having different inclinations on the front pin side and the rear pin side across the reference position Z ₀ (FIG. 4) are obtained. Even when the shape is asymmetrical, the in-focus position (Z _f ) can be detected accurately.

Furthermore, in the focus detection apparatus 10 of the present embodiment, when the peak shape is high, the focused position (Z _f ) is accurately detected in a short time by performing the simplified processing (normal calculation) in step S8. can do. Here, when the peak shape is high, it is possible to detect the in-focus position (Z _f ) by performing the process of step S7 (four-point calculation). However, when the peak ratio MAX / MIN is 1.05 or more, the processing of step S7 (4-point calculation) and the processing of step S8 (normal calculation) are performed with substantially the same accuracy. The in-focus position (Z _f ) can be detected. The detection error is about 10 to 20 μm, which is smaller than the thickness of the specimen 21. Therefore, when the peak ratio MAX / MIN is 1.05 or more, the in-focus position (Z _f ) can be accurately detected in a short time by performing the processing in step S8 (normal calculation).

In addition, since the focus detection apparatus 10 of the present embodiment uses a data string after one or more moving average processes (a high SN ratio from which high-frequency components have been sufficiently removed), the level of the peak shape is determined. Therefore, the index (peak ratio MAX / MIN) can be easily calculated. Furthermore, the in-focus position (Z _f ) can be easily calculated. In addition, by using the set value of the voltage applied to the photomultiplier tube (the sensitivity of the sensor unit), the number of executions of the moving average process can be determined easily and appropriately.

Furthermore, in the focus detection apparatus 10 of the present embodiment, in the process of Step S7 (four-point calculation), the interval D _{1 is set} to 60 μm and the interval D _{2 is set} to 120 μm, so that the reference position Z ₀ (FIG. 4) is interposed therebetween. Straight lines La and Lb having appropriate inclinations can be obtained on each of the front pin side and the rear pin side. Further, the movement range (main scan range) of the objective lens 23 determined in step S1 is also an appropriate interval (at least 240 μm or more (for example, about 300 μm)). Therefore, it is possible to accurately detect the in-focus position (Z _f ) even when the peak shape is low while keeping the time required for focus detection appropriately.

In the focus detection apparatus 10 of the present embodiment, the focus detection result (focus position (Z _f ) determined in step S11) is output from the focus position calculation circuit 14 to the position control circuit 15, and the position control circuit 15 In addition, by actually moving the objective lens 23 via the drive circuit 12 and the moving mechanism 24, high-accuracy autofocus is realized by weak fluorescence from the specimen 21.
(Modification)
In the above-described embodiment, the level of the peak shape is determined, and when the peak shape is low, the processing of step S7 (four-point calculation) is performed, but the present invention is not limited to this. The present invention can also be applied to the case where the process of step S7 (four-point calculation) is performed regardless of the height of the peak shape.

Further, in the above-described embodiment, the peak ratio MAX / MIN is used as the determination index of the peak shape, and the determination criterion is “1.05”, but the present invention is not limited to this. The determination criterion may be a value other than “1.05”. In determining the judgment criteria, it is preferable to consider the thickness of the specimen 21 and the allowable detection error.
Furthermore, in the above-described embodiment, when the peak shape is high, the process of step S6 (normal calculation) is performed, but the present invention is not limited to this. For example, the present invention can be applied to the case where the position of the objective lens 23 corresponding to the maximum intensity MAX of the light amount signal is determined as the in-focus position instead of the process of step S6.

In the above-described embodiment, the example in which the process proceeds to the process in step S9 after the process (normal calculation) in step S8 has been described, but the present invention is not limited to this. Since the calculation result in step S8 is always appropriate, the process may proceed to step S10 after step S8.
Furthermore, in the above-described embodiment, the intensity (minimum intensity MIN) of the light amount signal output from the photomultiplier tube of the light amount detection circuit 11 at the upper limit end and the lower limit end of the movement range of the objective lens 23 is the background component. Although the case where it is equal to intensity | strength was demonstrated, this invention is not limited to this. The present invention can also be applied to a case where the moving range of the objective lens 23 is set narrower than the above (minimum intensity MIN> background component intensity). At this time, the criterion for determining the peak shape is smaller than the above value (1.05).

In the embodiment described above, in the process of step S7 (4-point calculation), the interval D _{1 is set} to 60 μm and the interval D _{2 is set} to 120 μm. However, the present invention is not limited to this. The present invention can be applied if the interval D ₁ and the interval D ₂ are different values. The values of the interval D ₁ and the interval D ₂ may be changed according to the sample 21.
Furthermore, in the above-described embodiment, the example of the photomultiplier tube that collectively detects the amount of light incident on the light receiving surface has been described, but the present invention is not limited to this. For example, a configuration in which the amount of light is divided and detected, and the sum is obtained by a signal processing system at a later stage can be considered.

In the embodiment described above, the configuration in which the stage 22 of the specimen 21 is fixed and the objective lens 23 is moved in the vertical direction has been described as an example, but the present invention is not limited to this. The present invention can also be applied to the case where the objective lens 23 is fixed and the stage 22 is moved in the vertical direction.
Furthermore, in the above-described embodiment, the case has been described in which weak fluorescence generated from the object (specimen 21) for fluorescence observation is taken to perform focus detection, but the present invention is not limited to this. The present invention can also be applied when focus detection is performed by taking in weak light (for example, chemiluminescence of the specimen itself) generated from the specimen of the observation object.

It is a block diagram which shows the structure of the focus detection apparatus 10 of this embodiment. 3 is a flowchart illustrating a procedure of a detection operation of the focus detection device 10. It is a figure explaining an example of a matrix graph and a moving average process. It is a figure explaining 4 point type | formula calculation in case a peak shape is low. It is a figure explaining normal calculation in case a peak shape is high.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 Focus detection apparatus 11 Light quantity detection circuit 12 Drive circuit 13 Memory circuit 14 Focus position calculation circuit 15 Position control circuit 20 Case 21 Sample 22 Stage 23 Objective lens 24 Moving mechanism 25 Light source 26 Dichroic mirror 27 Half mirror 28 Predetermined imaging surface

Claims (5)

A sensor unit that detects light for focus detection generated from the observation object and outputs a light amount signal corresponding to a relative position between the object and the objective lens;
A generation unit that captures the light amount signals at a plurality of the relative positions from the sensor unit, and generates a data string representing a relationship between the relative position and the intensity of the light amount signal;
A first setting unit that obtains the relative position corresponding to the maximum intensity of the light amount signal based on the data string, and sets the relative position as a reference position;
In the direction in which the relative position approaches the reference position, a first position that is separated from the reference position by a first interval and a second position that is separated from the reference position by a second interval different from the first interval are set. And a third position that is separated from the reference position by the first interval and a fourth position that is separated from the reference position by the second interval in a direction in which the relative position is separated from the reference position. Two setting sections;
Of each point of the data string, an intersection of a straight line connecting two points corresponding to the first position and the second position and a straight line connecting two points corresponding to the third position and the fourth position is obtained. And a determining unit that determines the relative position at the intersection as the in-focus position. In the focus detection apparatus of the microscope according to claim 1,
A judgment unit for judging a peak shape when the data string is graphed;
The first setting unit, the second setting unit, and the determination unit, when the peak shape is flat as a result of the determination by the determination unit, respectively, A focus detection apparatus for a microscope, comprising: setting processing of a position, the second position, the third position, and the fourth position, and determination processing of the in-focus position. The focus detection apparatus for a microscope according to claim 2,
The determination unit determines that the peak shape is flat when the maximum intensity MAX of the light amount signal in the data string and the intensity MIN of the background component satisfy the following conditional expression:
MAX / MIN <1.05
The focus detection apparatus of the microscope characterized by the above-mentioned. In the focus detection apparatus of the microscope of any one of Claims 1-3,
The generating unit takes in the light amount signals at a plurality of the relative positions from the sensor unit, and generates the data string by performing a moving average process of the intensities of the plurality of light amount signals adjacent to the relative positions. Microscope focus detection device characterized by the above. The focus detection apparatus for a microscope according to claim 4,
The generation unit determines the number of executions of the moving average process based on a sensitivity setting value of the sensor unit.

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