JP5035600B2 - Biological microscope - Google Patents

Description

  The present invention relates to a biological microscope having an automatic focusing function of an observation optical system using a focus error signal.

  In a biological microscope intended for long-time observation, an autofocus function is necessary because the focus of an observation image is shifted due to a temperature change or the like.

  FIG. 4 is a block diagram showing an example of a conventional biological microscope having an autofocus function. As shown in the figure, the biological microscope uses the observation optical system 100 for magnifying and observing the sample placed on the preparation 1 with the objective lens 101 and the observation unit 103, and the focus error detection light Ld to determine the in-focus state. A focus error detection optical system 200 to detect, a dichroic mirror 300 that superimposes the focus error detection light Ld on the optical path of the observation light Ls, and a focus error signal Se obtained from the focus error detection optical system 200, and an automatic focus operation is performed. The control means 400 is comprised.

  The objective lens 101 is moved in the optical axis direction (Z direction) by the lens actuator 102, and the control means 400 drives the lens actuator 102 using the focus error signal Se as a feedback amount.

  In the focus error detection optical system 200, the focus error detection light Ld output from the focus error detection light source 201 passes through the lens 202, is reflected by the half mirror 203, and is superimposed on the optical path of the observation light Ls by the dichroic mirror 300. Then, the observation object (preparation 1) is irradiated.

  Further, only the focus error detection light Ld component of the return light (reflected light) from the observation target (preparation 1) is reflected by the dichroic mirror 300 and enters the focus error detection optical system 200. Incident light passes through the half mirror 203 and reaches the quadrant photodiode 206 via the collimator lens 204 and the cylindrical lens 205. The collimator lens 204, the cylindrical lens 205, and the quadrant photodiode 206 perform focus error detection by the astigmatism method and generate a focus error signal Se.

  The filters 104 and 207 are inserted so that the observation light Ls or the focus error detection light Ld does not enter another optical system.

  When the objective lens 101 is moved in the optical axis direction (Z direction) in the thus configured biological microscope, the focus error signal Se draws an S-curve as shown in FIG. The control means 400 performs the autofocus operation with the point where the focus error signal Se is zero or any constant value on the S-curve as the in-focus state.

  For example, in the example shown in the figure, the focus error signal Se becomes zero when the focus position of the focus error detection light Ld (observation light Ls) is positioned on the surface of the preparation 1, so when focusing on this surface, The control unit 400 drives the lens actuator 102 to control the position of the objective lens 101 so that the focus error signal Se becomes zero. Hereinafter, the surface to be focused is referred to as a focus reference surface 2.

JP-A-5-88072

In a biological microscope, an autofocus operation is often performed at the start of observation or during observation. Usually, the autofocus operation is performed with the sample 3 placed at the center of the microscope field of view.
In general, when the optical axis of the focus error detection optical system 200 is adjusted, the optical axis is adjusted to be the same as that of the observation optical system 100, so that the focus error detection light Ld is at the same position as the observation light Ls, that is, the microscope field of view. It will be irradiated to almost the center.

  For this reason, if an autofocus operation is performed with the sample 3 placed on the preparation 1, the focus error detection light Ld is also irradiated onto the sample 3, and is affected by the refractive index of the sample 3. Thus, the change in the focus error signal Se with respect to the movement of the objective lens 101 in the optical axis direction (Z direction) may cause a disturbance as shown in FIG. The relationship between the field of view 4 of the microscope and the position of the sample 3 is shown in FIG. 6A, and the state of the focus error signal Se at this time is shown in FIG.

As described above, when the sample 3 is in the center of the microscope visual field, the change in the focus error signal Se is disturbed, and an accurate autofocus operation cannot be performed or the autofocus operation becomes unstable.
In order to avoid this, as shown in FIG. 7, the sample 3 must be removed from the center of the microscope field of view 4 and an autofocus operation must be performed in the absence of the sample 3. The relationship between the field of view 4 of the microscope and the position of the sample 3 is shown in FIG. 7A, and the state of the focus error signal Se at this time is shown in FIG.

  An object of the present invention is to eliminate the drawbacks of the conventional apparatus as described above, and to realize a biological microscope capable of performing an autofocus operation with a simple configuration even when a sample is placed at the center of a microscope field of view. Is.

  It is another object of the present invention to realize a biological microscope capable of detecting and displaying whether or not the autofocus operation is performed at a position deviated from a sample with a simple configuration.

The biological microscope of the present invention is
A dichroic mirror that transmits the observation light emitted from the observation unit, reflects the focus error detection light emitted from the focus error detection light source, and superimposes the optical axis of the observation light and the focus error detection light on the same optical axis; A focus error detection optical system that irradiates the focus reference plane with the light superimposed by the dichroic mirror via the objective lens and receives the return light from the focus reference plane to detect the in-focus state on the focus reference plane In a biological microscope having
Tilt the optical axis from the focus error detecting light source with respect to the optical axis of the observation light by tilting the dichroic mirror, the position of the sample irradiation position of and shifted from the center of the field of view of the biological microscope focus error detection light An irradiation position changing means for removing from ,
A focus error detection state determination means for detecting presence or absence of disturbance of a focus error signal obtained from the focus error detection optical system;
Display means for displaying the presence or absence of disturbance of the focus error signal according to the determination result of the focus error detection state determination means,
The focus error detection state discriminating means samples a change in the focus error signal with respect to the movement of the objective lens in the optical axis direction and detects it as a high frequency component, and detects the disturbance of the focus error signal from the magnitude of the high frequency component. The presence or absence of
The display means displays whether or not the focus error signal is disturbed when the sample is positioned at the center of the microscope visual field during observation of the sample or at the start of observation.

  It has a holding part which supports the dichroic mirror at a fixed angle, and the change of the angle in the dichroic mirror is adjusted by the mounting angle of the holding part to the microscope frame.

  Thus, in the biological microscope of the present invention, the optical axis of the focus error detection light is tilted with respect to the optical axis of the observation light in the objective lens, and the irradiation position of the focus error detection light on the focus reference plane is from the center of the field of view of this microscope. Since they are shifted, even when the sample is placed at the center of the visual field of the microscope, the irradiation position of the focus error detection light can be removed from the position of the sample, and a reliable autofocus operation can be performed.

  Also, a focus error detection state determination unit that detects the presence or absence of disturbance in the focus error signal obtained from the focus error detection optical system, and displays the presence or absence of disturbance in the focus error signal according to the determination result of the focus error detection state determination unit Display means that can detect and display whether or not the autofocus operation is performed at a position deviated from the sample, and display the operation status of the autofocus operation to the operator of the microscope. I can inform you.

  Hereinafter, the biological microscope of the present invention will be described with reference to FIGS.

  FIG. 1 is a block diagram showing an embodiment of a biological microscope of the present invention. In the figure, the same components as those in FIGS. 4 to 7 are denoted by the same reference numerals.

  In the example shown in the figure, the dichroic mirror 300 is supported by the holding unit 301 and attached to the frame 105 of the microscope. Usually, the dichroic mirror 300 is fixed to the holding portion 301 at an angle of 45 °, and a spacer member 302 such as shim tape is interposed between the holding portion 301 and the frame 105 so that the angle of the dichroic mirror 300 is 45. It is tilted from a few mrad.

  In this way, as shown in FIG. 2, the optical axis of the focus error detection light Ld can be tilted with respect to the optical axis of the observation light Ls, and the irradiation position Pd is changed to the irradiation position Ps of the observation light Ls (microscope field of view). Center).

  Therefore, even when the sample is placed at the center of the visual field of the microscope, the irradiation position Pd of the focus error detection light Ld is removed from the position of the sample, and a reliable autofocus operation is performed without being affected by the refractive index of the sample. be able to.

  Note that, by changing the angle of the dichroic mirror 300, the optical axis of the focus error detection light Ld incident on the focus error detection optical system 200 also changes. In response to this change, the four-division photodiode 206 is changed. The position of is adjusted.

  In the description of FIG. 1, the case where the angle of the dichroic mirror 300 is changed according to the mounting angle of the holding unit 301 is illustrated, but the support angle of the dichroic mirror 300 in the holding unit 301 is directly changed without using the spacer member 302 or the like. You may make it do.

  Alternatively, the incident angle of the focus error detection light Ld with respect to the dichroic mirror 300 may be changed by changing the optical axis itself of the focus error detection optical system 200.

  In the biological microscope as described above, most of the focus error detection light Ld reflected from the reference focal plane 2 is reflected by the dichroic mirror 300 in the direction of the focus error detection optical system 200, and the rest is also cut by the filter 104. Therefore, the light does not enter the observation unit 103 and disturb the observation.

  In other words, the observation optical system 100 cannot see the focus error detection light Ld, and the focus error detection optical system 200 irradiates the focus error detection light Ld on any position on the reference focal plane to perform an autofocus operation. It is not possible to confirm whether or not

Therefore, it is preferable that there is a means that can surely irradiate the focus error detection light LD at a position off the sample 3 and confirm that the focus error signal Se is not disturbed.

  FIG. 3 shows a focus error detection state determination unit 500 that detects the presence or absence of disturbance in the focus error signal Se obtained from the focus error detection optical system 200, and a focus error signal according to the determination result of the focus error detection state determination unit 500. It is a block diagram which shows one Example of the display means 600 which displays the presence or absence of disorder in Se.

  The focus error detection state determination unit 500 includes an AD conversion unit 501, a memory unit 502, and a signal processing unit 503. The AD conversion unit 501 and the memory unit 502 sample the change of the focus error signal Se with respect to the movement of the objective lens 101 in the optical axis direction (Z direction) for a predetermined interval, and the signal processing unit 503 performs Fourier transform on the data string. Then, the presence or absence of disturbance of the focus error signal Se is determined from the magnitude of the high frequency component. That is, if there is disturbance in the focus error signal Se, the high frequency component becomes large, and when this exceeds a certain value, it is determined whether or not it is inappropriate for the autofocus operation and a discrimination output is generated.

  The display unit 600 includes a light emitting diode, and displays the determination result according to the output of the focus error detection state determination unit 500. For example, it is turned on when the focus error signal Se is disturbed, and is turned off when there is no disorder.

  Note that the above-described determination operation is performed together with the pull-in operation in which the objective lens 101 is scanned in the optical axis direction (Z direction) at the start of the automatic focus operation to obtain an S-shaped curve of the focus error signal Se. .

  Accordingly, when the sample 3 is positioned at the center of the microscope visual field at the start of observation or during observation, the sample 3 will be in focus error if the position of the sample 3 is adjusted while checking the display of the display means 600. It can be determined whether or not the irradiation position of the detection light Ld is applied, and the sample 3 is positioned at a position where the sample 3 can reliably perform the autofocus operation without affecting the focus error signal Se. can do.

  As described above, in the biological microscope of the present invention, the optical axis of the focus error detection light is tilted with respect to the optical axis of the observation light in the objective lens, and the irradiation position of the focus error detection light on the focus reference plane is set to the field of view of this microscope. Since the position is shifted from the center, even when the sample is placed at the center of the field of view of the microscope, the irradiation position of the focus error detection light is removed from the position of the sample, and a reliable autofocus operation can be performed.

  Also, a focus error detection state determination unit that detects the presence or absence of disturbance in the focus error signal obtained from the focus error detection optical system, and displays the presence or absence of disturbance in the focus error signal according to the determination result of the focus error detection state determination unit Display means that can detect and display whether or not the autofocus operation is performed at a position deviated from the sample, and display the operation status of the autofocus operation to the operator of the microscope. I can inform you.

  In the above description, the focus error detection means using the astigmatism method is exemplified as the focus error detection optical system 200, but the focus error detection method is not limited to this.

The block diagram which shows one Example of the biological microscope of this invention. The figure which shows the mode of the optical axis of the observation light Ls and the focus error detection light Ld. The block diagram which shows one Example of a focus error detection state determination means and a display means. The block diagram which shows an example of the conventional biological microscope. The figure which shows the mode of a focus error signal changing with respect to the movement of an objective lens. The figure which shows the mode of the change of the position of the sample in a microscope visual field, and a focus error signal. The figure which shows the mode of the change of the position of the sample in a microscope visual field, and a focus error signal.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Preparation 2 Focus reference plane 3 Sample 4 Field of view 100 Observation optical system 101 Objective lens 102 Lens actuator 103 Observation part 200 Focus error detection optical system 300 Dichroic mirror 400 Control means 500 Focus error detection state discrimination means 600 Display means

Claims (2)

A dichroic mirror that transmits the observation light emitted from the observation unit, reflects the focus error detection light emitted from the focus error detection light source, and superimposes the optical axis of the observation light and the focus error detection light on the same optical axis; A focus error detection optical system that irradiates the focus reference plane with the light superimposed by the dichroic mirror via the objective lens and receives the return light from the focus reference plane to detect the in-focus state on the focus reference plane In a biological microscope having
Tilt the optical axis from the focus error detecting light source with respect to the optical axis of the observation light by tilting the dichroic mirror, the position of the sample irradiation position of and shifted from the center of the field of view of the biological microscope focus error detection light An irradiation position changing means for removing from ,
A focus error detection state determination means for detecting presence or absence of disturbance of a focus error signal obtained from the focus error detection optical system;
Display means for displaying the presence or absence of disturbance of the focus error signal according to the determination result of the focus error detection state determination means,
The focus error detection state discriminating means samples a change in the focus error signal with respect to the movement of the objective lens in the optical axis direction and detects it as a high frequency component, and detects the disturbance of the focus error signal from the magnitude of the high frequency component. The presence or absence of
The biological microscope characterized in that the display means displays whether or not the focus error signal is disturbed when the sample is positioned at the center of the microscope visual field during observation of the sample or at the start of observation.   2. The biological microscope according to claim 1, further comprising a holding portion that supports the dichroic mirror at a constant angle, and the change of the angle of the dichroic mirror is adjusted by an angle of attachment of the holding portion to the microscope frame. .

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