JP5831782B2 - Microscope equipment - Google Patents

Description

  The present invention relates to a microscope apparatus.

  In recent years, microscopes with focus maintaining devices are expanding the market. A focus maintaining device that keeps focusing on the specimen at all times can effectively remove blurs and blurs of the observation image that accompany long-time time-lapse observation and reagent administration. One method for maintaining focus is an active method. In this method, the position of the specimen in the Z direction (the position of the objective lens in the optical axis direction) is detected from the reflection of focus light (hereinafter referred to as “AF light”) applied to the specimen (such as a cover glass). At this time, light having a wavelength different from that of the original illumination light / signal light (light from the specimen) of the microscope is used as AF light (see, for example, Patent Document 1). Then, the AF light detection unit removes light other than the AF light using the wavelength information, and detects only the AF light.

JP 2007-323094 A

  However, in the method of detecting AF light based on wavelength information, the wavelength of light used as AF light cannot be used as the wavelength of illumination light / signal light, and the degree of freedom of wavelength selectivity in microscope observation is reduced. There was a problem. For example, in a conventional fluorescence microscope, both illumination light and signal light (fluorescence) are in the visible range, so it is relatively easy to avoid the problem of wavelength overlap by using near infrared light as AF light. However, recently, a nonlinear optical microscope represented by a two-photon excitation fluorescence microscope has attracted attention. In these methods, the wavelength relationship between illumination light and signal light is very diverse, and it is very difficult to determine a wavelength region that does not overlap with AF light. Therefore, there is a need for an AF light detection mechanism that does not limit the wavelength selectivity of illumination light / signal light.

  The present invention has been made in view of such problems, and provides a microscope apparatus having a focus maintaining device capable of detecting only AF light even if the wavelength of AF light overlaps with the wavelength of signal light / illumination light. For the purpose.

In order to solve the above-described problem, a microscope apparatus according to the present invention has an objective lens, and irradiates the specimen with illumination light that is intensity-modulated at the first frequency via the objective lens . A microscope device that acquires fluorescence whose intensity changes at a frequency of 1, and that emits focus light that is intensity-modulated at a second frequency and has a wavelength that overlaps at least one wavelength region of illumination light and fluorescence. A light detection unit that detects light and outputs a detection signal; and irradiates the specimen with focus light through the objective lens, and is a position along the optical axis direction of the objective lens, and a predetermined position of the specimen A focus optical system that guides the focus light reflected at a position different from that to the light detection unit, a signal extraction unit that extracts a signal having the second frequency from the detection signal output from the light detection unit, and a signal extraction unit Based on the signal, by changing the relative positional relationship between the objective lens and the specimen, it has a focal maintenance device having a control unit for maintaining the focal plane of the objective lens at a predetermined position of the sample, a first The second frequency is lower than the first frequency.

In such a microscope apparatus , it is preferable that the first frequency is not an integral multiple of the second frequency.

In such a microscope apparatus , the light source unit outputs a light source that emits light, a signal generator that outputs a signal of the second frequency, and an intensity of light emitted from the light source from the signal generator. An intensity modulation element that modulates the intensity of the signal and emits the light as focus light, and the signal extraction unit includes a detection mechanism that extracts a signal having the second frequency from the detection signal by a signal output from the signal generator. It is preferable to have.

In such a microscope apparatus , the light source unit includes a signal generator that outputs a signal of the second frequency, and a light source that outputs focus light that is intensity-modulated by a signal output from the signal generator. The signal extraction unit preferably includes a detection mechanism that extracts a signal having the second frequency from the detection signal by a signal output from the signal generator.

Further, in such a microscope apparatus , the light source unit is extracted with the light source that emits the pulse light repeated at the second frequency as the focus light, the optical member that extracts a part of the focus light, and the optical member. And a signal extraction unit that extracts a signal having the second frequency from the detection signal by using the signal having the second frequency output from the photodetector. It is preferable to have a detection mechanism.

  By configuring the present invention as described above, it is possible to provide a microscope apparatus having a focus maintaining device capable of detecting only AF light even when the wavelength of AF light overlaps with the wavelength of signal light / illumination light.

It is explanatory drawing which shows the structure of the focus maintenance apparatus which concerns on 1st Embodiment. It is explanatory drawing which shows the relationship between the imaging surface of AF light by an objective lens, and a sample surface, Comprising: (a) shows the case where an imaging surface and a sample surface correspond, (b) shows a sample surface. The case where it exists in the back | inner side from an image formation surface is shown, (c) shows the case where a sample surface exists in a near side from an image formation surface. It is explanatory drawing which shows the relationship between AF light, illumination light, and signal light. It is explanatory drawing which shows the structure of the focus maintenance apparatus which concerns on 2nd Embodiment. It is explanatory drawing which shows the structure of the focus maintenance apparatus which concerns on 3rd Embodiment.

[First Embodiment]
Hereinafter, preferred embodiments of the present invention will be described with reference to the drawings. First, the configuration of the focus maintaining apparatus according to the present embodiment will be described. The focus maintaining apparatus 10 guides AF light from a light source unit 20 having a light source 21 such as an LED to an objective lens 61 of a microscope apparatus 60 using a focus optical system 30, and the specimen surface O is passed through the objective lens 61. 4, and the reflected light is a quadrant photodiode 40 (a photodetection element in which a circular region is divided into four, and a PD (photodiode) is arranged in each region, hereinafter referred to as “QPD40”) The position of the sample surface O with respect to the objective lens 61 is detected in the Z-axis direction by detecting with a light detection unit constituted by, for example.

  A focus optical system 30 of the focus maintaining apparatus 10 includes, in order from the light source 21 side, a collimator lens 31 that converts AF light emitted from the light source 21 into a substantially parallel light beam, a mask 32 that blocks approximately half of the substantially parallel light beam, and an incident light. The first half mirror 33 that transmits substantially half of the AF light to be reflected and reflects the rest, the offset lens 34 having a function to be described later, and the objective lens 61 that constitutes the microscope apparatus 60 are arranged on the image side. A second half mirror 35 is provided that guides the emitted AF light to the objective lens 61 and guides the AF light reflected from the sample surface O to the focus maintaining apparatus 10.

  Further, the focus optical system 30 of the focus maintaining apparatus 10 includes an imaging lens 36 in a direction in which AF light (reflected light) from the sample surface O is reflected by the first half mirror 33. The QPD 40 is disposed on the focal plane of the imaging lens 36. The light source unit 20 of the focus maintaining apparatus 10 outputs an acoustooptic element 22 (hereinafter referred to as “AOM22”) that is an intensity modulation element that modulates the intensity of AF light, and a signal that vibrates at a predetermined frequency fm. A function generator 23 (hereinafter referred to as “FG23”) that is a signal generator, and a driver 24 that controls the operation of the AOM 22 in accordance with a signal from the FG 23, and the focus maintaining apparatus 10 will be described later. The lock-in amplifier 41, which is a detection mechanism that constitutes a signal extraction unit that extracts a predetermined signal from the signal acquired by the QPD 40 in accordance with the signal from the FG 23, and the signal extracted by the lock-in amplifier 41 And a control unit 50 for controlling the focusing of the microscope apparatus 60 based on the above. As described above, in the first embodiment, the light source unit 20 that emits AF light is configured by the light source 21 and the AOM 22.

  In the focus maintaining apparatus 10 having such a configuration, the AF light emitted from the light source 21 is converted into a substantially parallel light beam by the collimating lens 31, and then the beam profile is made into a half-moon shape by the mask 32 and enters the AOM 22. The AOM 22 is controlled to be repeatedly turned on and off at a frequency fm by the FG 23 and the driver 24, and intensity modulation is performed on the AF light transmitted through the AOM 22 and emitted at the modulation frequency fm. Almost half of the AF light passes through the first half mirror 33, undergoes a change in curvature by the offset lens 34, is further combined with illumination light by the second half mirror 35, and is guided to the objective lens 61. Then, the AF light collected by the objective lens 61 is reflected by the sample surface O and passes through the objective lens 61 again. Thereafter, the light is reflected by the second half mirror 35, passes through the offset lens 34, is reflected by the first half mirror 33, and is condensed on the QPD 40 by the imaging lens 36.

  The offset lens 34 has a function of shifting the AF light imaging position in the Z-axis direction with respect to the focal plane of the objective lens 61. That is, when the AF light emitted from the light source 21 and made into a substantially parallel light beam by the collimator lens 31 is substantially parallel light after passing through the offset lens 34, the position of the AF light imaged by the objective lens 61 is the objective. This substantially coincides with the focal plane of the lens 61. On the other hand, when the AF light emitted from the offset lens 34 is divergent light, the image formation position of the AF light moves to the back side from the focal plane of the objective lens 61, and the AF light emitted from the offset lens 34 is convergent light. In some cases, the imaging position of the AF light moves to the near side from the focal plane of the objective lens 61. In this way, by shifting (offset) the imaging position of the AF light in the optical axis direction with respect to the focal plane of the objective lens 61, the focal point of the objective lens 61 is focused on a desired position in the sample. The focus can be maintained by reflecting the AF light at a position different from the focal plane of the objective lens 61 (for example, the boundary surface between the plate glass covering the specimen and the specimen (the above-described “specimen plane O”)).

  As shown in FIG. 2A, when the sample surface O is at the position designated by the offset lens 34, the light incident on the imaging lens 36 is substantially parallel light (in FIG. 2, the optical axis direction). In the plane perpendicular to the optical axis, one of the directions orthogonal to each other is defined as the X-axis direction and the other as the Y-axis direction). This represents a state where there is no focus shift, and light is imaged at the center of the QPD 40. On the other hand, as shown in FIGS. 2B and 2C, when the focus shift occurs, the light intensity distribution on the QPD 40 changes. For example, as shown in FIG. 2B, when the sample surface O is separated from the objective lens 61, the light incident on the imaging lens 36 becomes convergent light. Therefore, an image is formed in front of QPD 40 and is shifted to the left on QPD 40. Further, as shown in FIG. 2C, when the sample surface O approaches the objective lens 61, light incident on the imaging lens 36 becomes divergent light. For this reason, an image is formed behind the QPD 40 and shifted to the right on the QPD 40. In this way, the focus shift is detected as a positional shift on the QPD 40 by the knife edge method (half-moon focusing), and feedback control is performed by the control unit 50 so as to maintain the state of FIG. The focus can be maintained by moving the stage on which the sample is placed according to the detection result along the optical axis of the objective lens 61).

  At this time, illumination light and signal light are mixed in the light detected by each PD constituting the QPD 40 in addition to the AF light necessary for maintaining the focus as shown in FIG. Therefore, an electrical signal corresponding to the sum of the intensity of incident light is output from each PD. The output from the QPD 40 is sent to the lock-in amplifier 41. The lock-in amplifier 41 is a device that removes noise (in this case, signal light and illumination light) based on the frequency and phase information of a signal to be extracted (in this case, AF light). By taking the product of the output signal from the QPD 40 and the reference signal having the frequency fm, which is the same as that obtained by intensity-modulating the AF light output from the FG 23 with the AOM 22, the signal having the frequency fm among the signals output from the QPD 40 ( That is, only AF light) is converted into a DC component. This magnitude depends on the phase difference between the AF light and the reference signal. Then, only the AF light can be detected by detecting only the DC component by a low-pass filter (hereinafter referred to as “LPF”) built in the lock-in amplifier 41. This is because noise (signal light, illumination light) does not have a component of frequency fm, and thus is not converted into a direct current component and cannot pass through the LPF. In this way, the lock-in amplifier 41 performs synchronous detection only on a signal having the same frequency fm as the reference signal obtained from the FG 23 among the output signals from the QPD 40. Therefore, even if the wavelengths overlap, the AF light can be separated and detected from the illumination light / signal light. As described above, the signal (AF light) detected by the lock-in amplifier 41 has the focal position information of the specimen surface O, and the control unit 50 performs feedback processing using this signal, and the stage is displaced. Focus movement can be achieved by moving along the optical axis to correct.

  Here, considering the 1 / f fluctuation characteristics of noise, the higher the value of the frequency fm for modulating the AF light, the more noise the signal extracted by the lock-in amplifier 41 can be reduced. It is desirable to set it to 100 KHz or more. Further, the modulated AF light can be made more sensitive in the rectangular wave shape than in the sine wave shape.

  Further, in the microscope apparatus 60, when modulating the illumination light, the frequency for modulating the illumination light and the frequency fm for modulating the AF light must be set to different values. In view of the 1 / f fluctuation characteristics, in order to improve the sensitivity of the microscope body over the sensitivity of AF, it is better to lower the frequency fm for modulating the AF light than the frequency for modulating the illumination light. Thereby, even if a harmonic component due to modulation distortion occurs in the illumination light, it does not affect the AF light detection. At this time, if a harmonic component due to modulation distortion is generated in the AF light and the component matches or approximates the modulation frequency of the illumination light, the AF light lowers the detection sensitivity of the microscope body. For this reason, it is desirable that the frequency for modulating the illumination light is not an integral multiple of the frequency fm for modulating the AF light.

  As described above, the focus maintaining apparatus 10 according to this embodiment modulates the intensity of the AF light emitted from the light source 21 such as an LED with the AOM 22 at the predetermined frequency fm, thereby illuminating the AF light in the microscope apparatus 60. Even if the light or the signal light (fluorescence) has the same wavelength, only the AF light signal having a predetermined frequency fm can be extracted from the lock-in amplifier 41. There is no limit to the wavelength selection of light.

  The light source 21 is not limited to an LED, and an LD (laser diode) can also be used. The intensity modulation element may be an optical chopper or an electro-optic element instead of the acousto-optic element (AOM) 22. Further, a PD array can be used instead of the quadrant photodiode (QPD) 40.

[Second Embodiment]
In the first embodiment, the configuration of the light source unit 20 that modulates the intensity of the AF light emitted from the light source 21 such as an LED by the AOM 22 has been described. However, as shown in FIG. The light source unit 20 can be configured to modulate the intensity of the AF light emitted from the light source 21 by directly modulating the current injected into the semiconductor laser with the modulation frequency fm by the driver 24. According to such a configuration, modulated AF light can be used without using a modulation element such as the AOM 22 shown in the first embodiment. In FIG. 4, the same components as those in the first embodiment are denoted by the same reference numerals, and detailed description thereof is omitted (the same applies to the following).

  Further, when the modulation frequency of the light source 21 which is a semiconductor laser is increased, frequency chirp is generated and the wavelength of the AF light is changed, which may affect the observation of the specimen. Therefore, the modulation frequency fm at which no frequency chirp is generated. Is desirable.

[Third Embodiment]
In the first and second embodiments, the function generator (FG) 23 is provided in the light source unit 20 in order to modulate the AF light. However, as shown in FIG. This FG 23 is also unnecessary. The pulse laser emits pulsed light having a predetermined repetition frequency. By setting the repetition frequency to the above-mentioned predetermined frequency fm, the lock-in amplifier 41 can detect AF light having this frequency fm. it can. The reference signal input to the lock-in amplifier 41 is provided with an optical member 25 that reflects part of the AF light and transmits the other part of the AF light on the optical path of the AF light. It can be obtained by detecting with a photodiode (PD) 26 and outputting as a signal having a predetermined frequency. The optical member 25 may be any member that can reflect a part of AF light (about several percent) such as glass.

  In the case of the third embodiment, when a pulse laser is also used as the light source of illumination light, it is necessary that the repetition frequency of the illumination light pulse and the repetition frequency of the AF light pulse do not coincide with each other. There is. In addition, when performing excitation observation of a specimen, it is necessary that the intensity of the AF light is set so as not to generate fluorescence in the specimen (the intensity is not excited).

  In the first to third embodiments described above, the knife edge method (half-moon focusing) is shown as an example of the method for detecting the defocus, but the present invention is not limited to this method. Further, the presence or absence of the offset lens 34 does not limit the present invention.

DESCRIPTION OF SYMBOLS 10 Focus maintenance apparatus 20 Light source part 21 Light source 22 AOM (intensity modulation element)
23 Function generator (signal generator)
25 Optical element 26 Photodiode (photodetector)
30 Focus optical system 40 QPD (light detection unit)
41 Lock-in amplifier (detection mechanism) 50 Control unit 60 Microscope device 61 Objective lens

Claims (5)

A microscope apparatus having an objective lens, irradiating the specimen with illumination light whose intensity is modulated at the first frequency via the objective lens, and acquiring fluorescence from the specimen whose intensity changes at the first frequency Because
A light source unit that emits focus light having a wavelength that is intensity-modulated at a second frequency and overlaps at least one wavelength region of the illumination light and the fluorescence;
A light detection unit that detects light and outputs a detection signal;
The focus light is applied to the specimen through the objective lens, and the focus light reflected at a position along the optical axis direction of the objective lens, which is different from a predetermined position of the specimen. A focus optical system that leads to the light detector;
A signal extraction unit for extracting a signal having the second frequency from the detection signal output from the light detection unit;
A control unit that maintains a focal plane of the objective lens at a predetermined position of the sample by changing a relative positional relationship between the objective lens and the sample based on the signal extracted by the signal extraction unit; A focus maintaining device having
The microscope apparatus according to claim 1, wherein the second frequency is lower than the first frequency. The microscope apparatus according to claim 1, wherein the first frequency is not an integer multiple of the second frequency. The light source unit includes: a light source that emits light; a signal generator that outputs a signal of the second frequency; and an intensity of the light emitted from the light source by the signal output from the signal generator. An intensity modulation element that modulates and emits the focused light, and
The microscope according to claim 1, wherein the signal extraction unit includes a detection mechanism that extracts a signal having the second frequency from the detection signal based on the signal output from the signal generator. Equipment . The light source unit includes a signal generator that outputs a signal of the second frequency, and a light source that outputs focus light that is intensity-modulated by the signal output from the signal generator,
The microscope according to claim 1, wherein the signal extraction unit includes a detection mechanism that extracts a signal having the second frequency from the detection signal based on the signal output from the signal generator. Equipment . The light source unit emits pulsed light repeated at the second frequency as the focus light, an optical member that extracts a part of the focus light, and a part of the focus light extracted by the optical member A photodetector for detecting
2. The signal extracting unit includes a detection mechanism that extracts a signal having the second frequency from the detection signal based on the signal having the second frequency output from the photodetector. Or the microscope apparatus of 2.

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