JP2015001708A - Microscope system - Google Patents

Abstract

PROBLEM TO BE SOLVED: To easily generate a map image that includes information distributed in an optical axis direction of a focal plane of an objective lens in a specimen.SOLUTION: A microscope system 1 is provided that comprises: a motor-driven stage 11 that is capable of adjusting a position of a specimen; a scanner 17 that scans a laser beam; an objective lens 19 that condenses the laser beam on the specimen; an image acquisition part 21 that detects fluorescence from the specimen to acquire an image of the specimen; an acquisition position setting part 31 that sets an acquisition position of a partial image to be acquired by the image acquisition part 21 to the specimen; a beam shape change part 15 that is capable of changing a cross-sectional shape of the laser beam; a control part 33 that causes the motor-driven stage 11 and/or the scanner 17 to move a field of view range in accordance with the acquisition position set by the acquisition position setting part 31, and causes the image acquisition part 21 to acquire the partial image by changing the cross-sectional shape of the laser beam in a direction where depth of field is expanded by the beam shape change part 15; and a map image generation part 35 that generates a map image by arraying the partial image for each acquisition position.

Description

  The present invention relates to a microscope system.

  2. Description of the Related Art Conventionally, a microscope system that generates a wider field of view image, that is, a so-called map image by combining a plurality of adjacent partial images is known (see, for example, Patent Document 1). The microscope system described in Patent Document 1 can determine whether or not a sample exists from the synthesized map image even when the sample existing position is distributed in the optical axis direction of the focal position of the objective lens. Therefore, the diameter of the pinhole is increased, and fluorescence emitted from the optical axis other than the focal plane of the objective lens in the sample is also detected to acquire a partial image.

JP 2010-85420 A

  However, in the conventional microscope system, since the laser density per unit area is low in the portion of the focal position of the objective lens that moves back and forth in the optical axis direction, there is a possibility that there may be a region where the fluorescent dye cannot be excited. In addition, since multiphoton excitation does not occur other than the focal plane of the objective lens, it is not possible to detect fluorescence emitted from the optical axis other than the focal plane of the objective lens in the sample by multiphoton excitation. There is an inconvenience.

  The present invention has been made in view of the above-described circumstances, and provides a microscope system that can easily generate a map image including information distributed in the optical axis direction of the focal plane of an objective lens in a sample. Objective.

In order to solve the above problems, the present invention employs the following means.
The present invention includes a motorized stage on which a sample is mounted and the position of the sample can be adjusted, a scanner that scans a laser beam applied to the sample mounted on the motorized stage, and scanned by the scanner An objective lens that focuses laser light on the sample, an image acquisition unit that detects fluorescence generated in the sample by irradiation of laser light from the objective lens, and acquires an image of the sample; and Set by an acquisition position setting unit that sets an acquisition position of a partial image acquired by dividing the image by a predetermined visual field range by an image acquisition unit, a shape changing unit that can change a cross-sectional shape of laser light, and the acquisition position setting unit The laser beam is moved in the direction in which the visual field range is moved by the electric stage and / or the scanner in accordance with the acquired acquisition position and the depth of field is expanded by the shape changing unit. A microscope system comprising: a control unit that changes a cross-sectional shape to acquire the partial image by the image acquisition unit; and a map image generation unit that generates a map image by arranging the partial images acquired for each acquisition position. I will provide a.

  According to the present invention, when the sample is placed on the electric stage, the acquisition position of the partial image is set by the acquisition position setting unit, and the control unit moves the visual field range according to the acquisition position by the electric stage and / or the scanner. Let Then, at each acquisition position, the laser beam is scanned by the scanner and irradiated onto the sample by the objective lens, and the fluorescence from the sample is detected by the image acquisition unit to acquire a partial image. Thereby, the map image generation unit arranges the partial images for each acquisition position, and generates a map image of the sample over a wider visual field range.

  In this case, the control unit changes the cross-sectional shape of the laser light in the direction in which the depth of field is expanded by the shape changing unit when acquiring the partial image, so that the image acquiring unit emits from the focal plane of the objective lens. The partial image can be acquired by detecting not only the fluorescence emitted but also the fluorescence emitted from the optical axis other than the focal plane of the objective lens. As a result, a map image including information distributed in the optical axis direction of the focal plane of the objective lens can be generated.

In the above-described configuration, the shape changing unit may be an optical member that emits light by reducing the light beam diameter of the incident laser light and increasing the density of the light beam center.
With this configuration, it is possible to suppress a decrease in resolution in the direction intersecting the optical axis of the laser light, reduce the numerical aperture, and widen the depth of field.

In the above-described configuration, the shape changing unit may be a mechanical aperture member that limits the diameter of the laser beam that passes therethrough.
With this configuration, although the resolution in the direction intersecting the optical axis of the laser light is reduced, the numerical aperture can be reduced and the depth of field can be extended to a wider range.

In the above configuration, the shape changing unit may be an axicon prism that causes a laser beam having an annular light flux shape to enter the pupil position of the objective lens.
With this configuration, the resolution in the direction intersecting the optical axis of the laser light can be increased, and the depth of field can be increased by increasing the focal length of the laser light.

In the above configuration, the shape changing unit may be a phase modulation member that modulates the phase of the laser beam to generate a Bessel-shaped laser beam on the sample.
With this configuration, the resolution in the direction intersecting the optical axis of the laser light can be increased, and the depth of field can be increased by increasing the focal length of the laser light.

  According to the present invention, it is possible to easily generate a map image including information distributed in the optical axis direction of the focal plane of the objective lens in the sample.

1 is a schematic configuration diagram of a microscope system according to an embodiment of the present invention. It is a figure which shows the observation range of the sample displayed on a monitor, and the acquisition position of a partial image.

Hereinafter, a microscope system according to an embodiment of the present invention will be described with reference to the drawings.
As shown in FIG. 1, the microscope system 1 according to the present embodiment includes a laser scanning microscope 3 and a controller 5 that controls the laser scanning microscope 3.

  The laser scanning microscope 3 includes an electric stage 11 on which a sample (not shown) is placed, a light source 13 that generates laser light, and a beam shape changing unit that can change the cross-sectional shape of the laser light emitted from the light source 13. (Shape changing unit) 15, a scanner 17 that scans the laser light that has passed through the beam shape changing unit 15, and an objective lens that irradiates the sample with the laser light scanned by the scanner 17 and collects fluorescence generated in the sample 19, a dichroic mirror (not shown) that branches the fluorescence condensed by the objective lens 19 from the optical path of the laser light, and an image acquisition unit 21 that detects the fluorescence branched by the dichroic mirror and acquires an image of the sample. I have.

  The electric stage 11 includes three motors (not shown) and can move independently along movement axes in the X, Y, and Z directions orthogonal to each other. Thereby, the electric stage 11 can move the placed sample in a three-dimensional direction.

  The beam shape changing unit 15 is, for example, a beam expander (optical member), and emits the light beam diameter of the incident laser light by optically narrowing it. By narrowing the beam diameter of the laser beam by the beam expander, the density at the center of the beam can be increased to increase the focal length and the depth of field can be increased. In addition, since the skirt portion where the intensity of the Gaussian distribution of the laser beam is weak fills the pupil of the objective lens 19, the direction (X, Y) intersecting the optical axis of the laser beam compared to before changing the sectional shape of the laser beam. Direction) resolution can be suppressed. Further, since the density of the center of the light beam of the laser light incident on the pupil of the objective lens 19 is increased, it is strong against scattering.

  The scanner 17 is, for example, a proximity galvanometer mirror, and is configured by opposing two galvanometer mirrors (not shown) that can swing around mutually orthogonal axes. This scanner 17 can scan the laser beam two-dimensionally on the sample by swinging two galvanometer mirrors and deflecting the laser beam.

  The controller 5 acquires a partial image by the acquisition position setting unit 31 that sets the acquisition position of the partial image acquired by dividing the sample image, and by the image acquisition unit 21 at the acquisition position set by the acquisition position setting unit 31. A control unit 33 that controls the laser scanning microscope 3 and a map image generation unit 35 that generates a map image by arranging the partial images acquired for each acquisition position are provided.

  Further, a monitor 37 as shown in FIG. 2 is connected to the controller 5 so that the image acquired by the image acquisition unit 21, the image generated by the map image generation unit, and the like can be displayed on the monitor 37. It has become.

  The acquisition position setting unit 31 sets an observation range K in which an image of the sample is acquired as shown in FIG. 2 in accordance with an instruction input by a user through an input unit (not shown). In addition, the acquisition position setting unit 31 divides the set observation range K into a plurality of divided regions for each field of view of the objective lens 19, and acquires a plurality of acquisition positions P at which the partial image is acquired by the image acquisition unit 21. Is set as.

  The observation range K and the acquisition position P set by the acquisition position setting unit 31 are displayed on the sample image on the monitor 37. In addition, when the acquisition position P of the partial image is set by the acquisition position setting unit 31, coordinate information of each acquisition position P is sent to the map image generation unit 35.

  The control unit 33 moves the visual field range by the electric stage 11 and the scanner 17 in accordance with the acquisition position P set by the acquisition position setting unit 31. The movement order of the acquisition position P, that is, the acquisition order of partial images may be set by the user or may be set by the acquisition position setting unit 31.

  Further, when the image acquisition unit 21 acquires a partial image at each acquisition position P, the control unit 33 changes the cross-sectional shape of the laser light in the direction in which the beam shape changing unit 15 increases the depth of field. ing.

  The map image generation unit 35 arranges the partial images at the respective acquisition positions P acquired by the image acquisition unit 21 according to the respective coordinate information, and generates a two-dimensional map image of the entire observation range.

The operation of the microscope system 1 configured as described above will be described.
In order to acquire a map image of a sample by the microscope system 1 according to the present embodiment, first, the user places the sample on the electric stage 11, operates the controller 5, and instructs the observation range K of the sample from the input unit. To do.

  In the controller 5, the acquisition position setting unit 31 sets an observation range K of the sample in accordance with a user instruction as shown in FIG. Then, the acquisition position setting unit 31 divides the set observation range K for each predetermined visual field range, and sets a plurality of acquisition positions P of partial images.

  Next, the electric stage 11 and the scanner 17 are controlled by the control unit 33, and the visual field range is moved according to the initial acquisition position P of the partial image. Then, the laser light emitted from the light source 13 is changed by the beam shape changing unit 15 in the direction in which the depth of field is expanded, then deflected by the scanner 17 and irradiated onto the sample by the objective lens 19. Thereby, the laser beam is scanned two-dimensionally on the sample according to the swinging motion of the scanner 17.

  When fluorescence is generated in the sample by being irradiated with the laser light, the fluorescence is collected by the objective lens 19, branched by the dichroic mirror via the scanner 17, and detected by the image acquisition unit 21. Thereby, in the image acquisition part 21, the two-dimensional partial image of the initial acquisition position P of a sample is acquired.

  Subsequently, the electric stage 11 and the scanner 17 are controlled by the control unit 33, and the visual field range is moved in accordance with the second acquisition position P of the partial image. Then, similarly to the first acquisition position P, the laser light emitted from the light source 13 is two-dimensionally scanned, and the fluorescence from the sample is detected by the image acquisition unit 21. A partial image is acquired.

  In this manner, the laser scanning microscope 3 is controlled by the control unit 33, and partial images are acquired at each acquisition position P of the partial image set by the image acquisition position setting unit 31.

  Next, the operation of the map image generation unit 35 arranges the partial images of all the acquisition positions P acquired by the image acquisition unit 21 according to the respective coordinate information, and generates a two-dimensional map image of the entire observation range. . Thereby, a sample can be observed with the map image over a wider visual field range.

In this case, when the control unit 33 acquires a partial image at each acquisition position P, the beam shape changing unit 15 changes the cross-sectional shape of the laser light in the direction of widening the depth of field, whereby the image acquiring unit 21, not only the fluorescence emitted from the focal plane of the objective lens 19 but also the fluorescence emitted from the optical axis other than the focal plane of the objective lens 19 can be detected to obtain a partial image.
Therefore, according to the microscope system 1 according to the present embodiment, a map image including information distributed in the optical axis direction of the focal plane of the objective lens 19 can be easily generated.

This embodiment can be modified as follows.
In the present embodiment, the beam shape changing unit 15 has been described by exemplifying a beam expander. However, a lens member is not used, but a combination of concave mirrors having different focal lengths (optical member) may be used.

  The first modification may employ a mechanical diaphragm (mechanical diaphragm member) that limits the beam diameter of the laser light when passing the laser light emitted from the light source 13 as the beam shape changing unit.

  By using the mechanical aperture to cut the outer portion in the radial direction of the laser beam and reducing the numerical aperture, the focal length can be increased and the depth of field can be increased. In this case, although the resolution in the X and Y directions intersecting the optical axis of the laser beam is reduced, the depth of field can be further increased and the influence of scattering is strong.

  The second modification may employ an axicon prism that modulates the phase of the laser beam and causes the annular laser beam to be incident on the pupil position of the objective lens 19 as the beam shape changing unit. . In this case, an axicon prism in which a pair of conical prisms having the same refractive index and the same apex angle are arranged so that their optical axes coincide with each other and their vertices face each other may be adopted.

  With such an axicon prism, a laser beam having a cylindrical light beam shape emitted from the light source 13 is incident on one prism, and is converted into parallel light having a ring-shaped light beam shape from the other prism and emitted. Thus, it is possible to extend the depth of field by increasing the focal length. For example, the resolution in the direction intersecting the optical axis of the laser light can be increased and the depth of field can be further expanded compared to before changing the cross-sectional shape of the laser light.

  The third modification employs LCOS (Liquid Crystal On Silicon, a phase modulation member) as a beam shape changing unit, and modulates the phase of the laser beam to generate a Bessel-shaped laser beam on the sample. Also good. In this case, the LCOS may be arranged at a position conjugate with the pupil position of the objective lens 19.

  By using LCOS to change the wavefront shape of the laser light emitted from the light source 13 by phase modulation to generate a Bessel-shaped laser light on the sample, the depth of field can be increased by increasing the focal length.

As described above, according to the present invention, unlike the conventional method of acquiring the fluorescence generated from the optical axis other than the focal plane by opening the pinhole, by increasing the illumination-side spot, the efficiency can be improved even in the area other than the focal plane. Fluorescence can be excited automatically, and a map image obtained by acquiring fluorescence emitted from the optical axis other than the focal plane can be easily created.
In particular, in the case of multiphoton excitation, since fluorescence is generated only in a region where the photon density is extremely high, the photon density in regions other than the focal plane is increased by increasing the spot on the illumination side as in the present invention. This is effective because fluorescence can be generated by photon excitation.

DESCRIPTION OF SYMBOLS 1 Microscope system 11 Electric stage 15 Beam shape change part (shape change part)
DESCRIPTION OF SYMBOLS 17 Scanner 19 Objective lens 21 Image acquisition part 31 Acquisition position setting part 33 Control part 35 Map image generation part P Acquisition position

Claims (5)

A motorized stage on which a sample is placed and the position of the sample can be adjusted;
A scanner that scans a laser beam applied to the sample placed on the electric stage;
An objective lens for condensing the laser beam scanned by the scanner onto the sample;
An image acquisition unit for detecting fluorescence generated in the sample by irradiation of laser light from the objective lens and acquiring an image of the sample;
An acquisition position setting unit that sets an acquisition position of a partial image acquired by dividing the sample for each predetermined visual field range by the image acquisition unit;
A shape changer capable of changing the cross-sectional shape of the laser beam;
The field of view range is moved by the electric stage and / or the scanner in accordance with the acquisition position set by the acquisition position setting unit, and the cross-sectional shape of the laser light is increased in the direction of expanding the depth of field by the shape changing unit. A control unit for changing and acquiring the partial image by the image acquisition unit;
A microscope system comprising: a map image generation unit that generates a map image by arranging the partial images acquired for each acquisition position.   2. The microscope system according to claim 1, wherein the shape changing unit is an optical member that optically narrows an incident laser beam and emits it with an increased density at the center of the beam.   The microscope system according to claim 1, wherein the shape changing unit is a mechanical aperture member that limits a beam diameter of laser light passing therethrough.   2. The microscope system according to claim 1, wherein the shape changing unit is an axicon prism that causes laser light having an annular light flux shape to enter a pupil position of the objective lens.   The microscope system according to claim 1, wherein the shape changing unit is a phase modulation member that modulates a phase of laser light to generate a Bessel-shaped laser light on the sample.

Images