JP6357317B2 - Microscope illumination device, microscope illumination method, and microscope - Google Patents

Description

  The present invention relates to a microscope illumination device, a microscope illumination method, and a microscope.

2. Description of the Related Art Conventionally, a microscope using evanescent illumination is known as a technique for observing a specimen in an extremely thin range near the surface of a cover glass (see, for example, Patent Document 1).
The evanescent illumination causes the laser light collected on the lens edge at the back focal position of the immersion objective lens to enter the immersion objective lens, and the laser light that has become substantially parallel light by the immersion objective lens and the cover glass. In this illumination method, evanescent light is oozed out to the cover glass side by total reflection at the interface with the liquid. Although this is a general microscope technique that enables observation of a very thin specimen in the vicinity of the surface of the cover glass with high contrast, there is a problem that only partial fluorescence information can be obtained with total reflection illumination that illuminates from one direction.

  Therefore, by rotating the incident direction of the laser light on the cover glass around the axis of the immersion objective lens and acquiring a time-averaged image, it is possible to reduce shadow formation and speckle noise in the image by oblique illumination. A technique for suppressing the above is shown (for example, see Patent Document 2).

JP 2001-272606 A JP 2003-131141 A

  However, when the NA of the laser light incident on the back focal position of the immersion objective lens is increased in order to widen the field of view, the energy density of the laser light at the back focal position becomes extremely high, and the immersion objective is There is a disadvantage that the lens is damaged by heat. That is, if the NA of the laser beam is increased to widen the illumination field, the spot diameter of the laser beam formed at the back focal position becomes small and the brightness of the wide illumination field is made equal to that before spreading. Therefore, it is necessary to make high-energy laser light incident to that extent, and there is a disadvantage that the energy density of the laser light at the back focal position increases synergistically. Furthermore, since time averaging is required, it is impossible to photograph at high speed, and there is a disadvantage that subject displacement occurs due to vibration of a galvanometer mirror, a motor, or the like.

  The present invention has been made in view of the above-described circumstances, and is a microscope illumination apparatus and microscope that can expand a visual field range while preventing damage caused by heat of a lens that condenses laser light and performs Kohler illumination. An object is to provide an illumination method and a microscope.

In order to achieve the above object, the present invention provides the following means.
One aspect of the present invention, is incident laser light emitted from the light source, organic vital to and NA has a main optical axis distributed on a conical surface extending at an angle to the incident optical axis A light conversion unit that converts and emits laser light including light that has traveled along the incident optical axis, and a power that deflects the laser light emitted from the light conversion unit in a direction parallel to the incident optical axis. And a microscope illumination device that condenses laser light emitted from the optical element at a pupil position of a condenser lens for illuminating a specimen or a conjugate position thereof.

  According to this aspect, when the laser light emitted from the light source is incident on the light conversion unit, the laser light has the main optical axis distributed on the conical surface in the light conversion unit and is along the main optical axis. It is converted into laser light having NA to be condensed. The laser beam emitted from the light conversion unit is deflected by passing through the optical element, and becomes a laser beam having an NA in which the main optical axis is distributed on a cylindrical surface parallel to the incident optical axis.

  Then, by condensing such laser light at the pupil position of the condenser lens or its conjugate position, the laser light condensed by the condenser lens illuminates the sample as Koehler illumination. In this case, since the main optical axis is distributed in the circumferential direction around the optical axis of the condenser lens, the laser light incident on the condenser lens is incident on the specimen simultaneously from a plurality of circumferential directions. Illuminate.

  Therefore, the problem due to the oblique illumination is solved, and the condensing position of the laser light at the pupil position of the condensing lens is dispersed. Therefore, the NA of the laser light is increased and a laser beam having a large energy is introduced. However, the energy density at the condensing position does not become excessive, and the condensing lens can be maintained in a healthy state. Furthermore, since time averaging is not required, high-speed imaging is possible, and no vibration is generated, so that subject blurring does not occur.

  Further, since the optical element has no power, the NA of the laser light is stored without change before and after passing through the optical element. That is, the cylindrical surface on which the main optical axis of the laser beam emitted from the optical element is arranged depending on where the optical element is arranged on the optical axis that is spreading at a predetermined angle with respect to the incident optical axis The angle of the Koehler illumination that is incident on the sample by the condenser lens can be continuously changed, and an optimal illumination state can be provided for the objective lens, the sample, and the observation conditions. In this case as well, since the NA of the laser light does not change, the beam diameter of the Koehler illumination can be kept constant, and the brightness in the illumination field can be kept constant.

In the above aspect, the light conversion unit may be a diffraction grating that deflects laser light and imparts NA.
By doing so, the energy density of the laser beam at the pupil position of the condenser lens can be easily dispersed by a single diffraction grating, and the visual field range is expanded while preventing damage to the condenser lens that performs Koehler illumination. be able to.

Moreover, in the said aspect, the said light conversion part may be provided with the lens which has a positive power which provides NA to a laser beam, and the diffraction grating which deflects a laser beam.
In this way, the function of the diffraction grating can be simplified and simplified. The laser light after NA is given by the lens may be deflected by the diffraction grating, or vice versa.

Moreover, in the said aspect, the said light conversion part may convert into the laser beam distributed continuously over the perimeter on the said conical surface.
By doing in this way, the energy density of the laser beam condensed to the pupil position of a condensing lens can be made the lowest, and a wide and bright visual field range can be ensured.

Moreover, in the said aspect, the said optical element may be provided so that a movement is possible along the incident optical axis direction of the said laser beam.
By doing in this way, the incident position of the laser beam to the pupil position of the condensing lens in the radial direction can be changed, and the incident angle of the Koehler illumination to the sample by the condensing lens can be continuously adjusted. In this case, the brightness of the illumination field can be maintained constant even when the incident angle is changed.

Another aspect of the present invention, a laser beam emitted from the light source, have a and NA has a main optical axis distributed on a conical surface extending at an angle to the incident optical axis A first step of converting the laser light including light that has traveled along the incident optical axis; and the laser light converted in the first step is parallel to the incident optical axis by an optical element having no power. A second step of deflecting in the direction; a third step of condensing the laser light deflected in the second step at a pupil position of a condenser lens for illuminating the specimen or a conjugate position thereof; And a fourth step of moving the optical element along an incident optical axis direction of the laser light.

  According to another aspect of the present invention, there is provided a stage on which a sample is mounted, a condensing lens arranged to face the sample mounted on the stage, and laser light incident on the condensing lens. A microscope comprising the microscope illumination device according to claim 5 is provided.

  According to the present invention, it is possible to expand the visual field range while preventing damage caused by heat of a lens that performs Koehler illumination by condensing laser light.

It is a schematic diagram which shows the structure of the microscope which concerns on one Embodiment of this invention. In the microscope of FIG. 1, it is a figure which shows an example of the condensing pattern of (a) laser beam of arrow AA, and (b) another example, respectively. It is a schematic diagram which shows the change of the shape of the light beam when an axicon lens is moved to an optical axis direction in the microscope of FIG. It is a flowchart explaining the microscope illumination method which concerns on one Embodiment of this invention. It is a schematic diagram which respectively shows the microscope illumination apparatus which concerns on (a) this embodiment of the microscope of FIG. 1, and the microscope illumination apparatus which concerns on (b) modification. It is a schematic diagram which shows the microscope illuminating device which concerns on the other modification of the microscope of FIG.

A microscope illumination device 4, a microscope 1, and a microscope illumination method according to an embodiment of the present invention will be described below with reference to the drawings.
As shown in FIG. 1, a microscope 1 according to this embodiment includes a stage 2 on which a specimen S is mounted, and an immersion objective lens (condensing lens) 3 that is disposed close to the lower side of the stage 2 and facing upward. And a microscope illumination device 4 that irradiates the specimen S with laser light through the immersion objective lens 3.

  The specimen S is mounted on the stage 2 while being placed on the cover glass G. The stage 2 is provided with an opening 2a, and the laser beam condensed by the immersion objective lens 3 is irradiated from below through the cover glass G on the stage 2 through the opening 2a. ing.

  Although the immersion objective lens 3 is represented by a single lens in the drawing, it is actually configured by combining a number of lenses. A liquid B such as pure water is injected into the gap between the upper surface 3a of the most advanced lens of the immersion objective lens 3 and the cover glass G, and is held in the gap by surface tension.

The microscope illumination device 4 according to the present embodiment includes a collimating lens 6 that condenses the laser light L1 from the light source guided by the optical fiber 5 and converts it into laser light L2 that is substantially parallel light, and the collimating lens. 6 includes a diffraction grating 7 that diffracts the laser light L2 that has been converted into substantially parallel light by 6 and transmits the laser light L3 emitted from the diffraction grating 7 and that has no power and an axicon lens 8 that has no power. ing.
In the figure, reference numerals 9 and 10 are relay lenses that relay the pupil position P of the immersion objective lens 3, reference numeral 11 is an aperture, and reference numeral 12 is a mirror.

  The diffraction grating 7 is distributed on a conical surface that spreads at a predetermined angle with respect to the incident optical axis, as shown in FIG. 1, when the laser beam L2 composed of substantially parallel light is incident and transmitted. The laser beam L3 having a main optical axis and having an NA is converted and emitted. That is, as shown in FIG. 2A, the laser beam L3 diffracted by the diffraction grating 7 is condensed at the condensing position with an annular pattern C continuous in the circumferential direction. It has become.

  In the present embodiment, in the diffraction grating 7, as shown in FIG. 2A, the laser beam L <b> 3 is collected in a pattern C that is continuous in the circumferential direction, but is continuous. However, it is not always necessary, and as shown in FIG. 2 (b), the laser beam L3 may be converted into a pattern C ′ that is focused at a number of positions at intervals in the circumferential direction. The number of spots may be an arbitrary number of 2 or more, and the larger the number, the more effective.

  The axicon lens 8 has a tapered surface 8a on the incident end side. The inclination angle of the taper surface 8 a is set to an angle that deflects the main optical axis of the laser light L 3 emitted from the diffraction grating 7 in a direction parallel to the incident optical axis to the diffraction grating 7. Since the axicon lens 8 has no power, the NA of the laser beams L3 and L4 does not change before and after passing through the axicon lens 8.

  The diffraction grating 7 and the axicon lens 8 are arranged so that the condensing position of the laser light L4 emitted from the axicon lens 8 coincides with the rear focal position Q of the rear lens 9 constituting the relay lenses 9 and 10. Is arranged. In other words, the laser beam L4 emitted from the axicon lens 8 is condensed at a position Q optically conjugate with the pupil position P of the immersion objective lens 3.

  Further, the axicon lens 8 is provided so as to be movable in a direction along the optical axis of incidence of the laser light L2 on the diffraction grating 7. When the axicon lens 8 is moved in this direction, as shown in FIG. 3, the incident position of the laser light L3 emitted from the diffraction grating 7 on the tapered surface 8a of the axicon lens 8 changes in the radial direction. The diameter of the pattern at the condensing position of the laser beam L4 changes. In the example shown in FIG. 3, the radial dimension of the circular pattern at the condensing position Q of the laser beam L4 is reduced by moving the axicon lens 8 in a direction close to the diffraction grating 7.

A microscope illumination method using the microscope illumination device 4 and the microscope 1 according to the present embodiment configured as described above will be described below.
In order to observe the specimen S using the microscope 1 according to the present embodiment, the specimen S placed on the cover glass G is mounted on the stage 2 and the laser light L1 from the light source is emitted from the exit end of the optical fiber 5. .

In the microscope illumination method according to the present embodiment, as shown in FIG. 4, laser light L <b> 2 emitted from the emission end of the optical fiber 5 and converted into substantially parallel light by the collimator lens 6 is incident on the incident light by the diffraction grating 7. A first step S1 for converting into a laser beam L3 having a main optical axis distributed on a conical surface extending around the axis and having NA, and the laser beam L3 converted in the first step S1 A second step S2 that deflects in a direction parallel to the incident optical axis by the axicon lens 8 that does not include the laser beam L4 deflected in the second step S2 and the pupil position P of the immersion objective lens 3; A third step S3 for condensing light at the conjugate position Q and a fourth step S4 for moving the axicon lens 8 along the incident optical axis direction are included.

  The laser light L1 emitted from the emission end of the optical fiber 5 is converted into laser light L2 composed of substantially parallel light by the collimator lens 6 and then incident on the diffraction grating 7 in the first step s1. The laser beam L3 is diffracted by the diffraction grating 7, thereby having an annular pattern having a main optical axis extending on a conical surface with the incident optical axis as the center, and being focused by a predetermined NA. Is converted to Thereafter, the laser light L3 is transmitted through the axicon lens 8 in the second step S2, so that the main optical axis is deflected in a direction parallel to the incident optical axis, and on the cylindrical surface with the incident optical axis as the center. The laser beam L4 having an annular pattern having a main optical axis extending in the direction is condensed at the rear focal position Q of the relay lens 9 in the third step S3.

  The condensed laser light L4 is relayed by the relay lenses 9 and 10, deflected by 90 ° by the mirror 12, and condensed at the pupil position P which is the back focal position of the immersion objective lens 3. As a result, the laser beam L5 collected by the immersion objective lens 3 propagates through the liquid B as parallel light and is obliquely incident on the bottom surface of the cover glass G from the entire circumference direction of the immersion objective lens 3. . When the incident angle of the laser beam L5 is shallower than the critical angle, the laser beam L5 is totally reflected at the interface between the cover glass G and the liquid B, and the evanescent light passes through the cover glass G and passes through the cover glass G. A very thin region in the vicinity of the bonding surface of the specimen S bonded to the upper surface is illuminated.

As a result of the irradiation with the evanescent light, the fluorescence generated in the vicinity of the adhesion surface of the specimen S is collected by the immersion objective lens 3 and is not shown (for example, disposed on the specimen side with respect to the axicon lens 8). The fluorescent image can be obtained by separating and photographing from the laser beam L5.
In this case, according to the microscope illuminating device 4 according to the present embodiment, the laser light L4 is dispersed in a ring shape by the diffraction grating 7, so the laser light L4 at the pupil position P of the immersion objective lens 3 is at one point. Dispersed over the entire circumference without concentration.

  Thereby, in order to increase the visual field range, the NA of the laser light L4 incident on the pupil position P of the immersion objective lens 3 is increased, so that the spot diameter at the pupil position P is further expanded even if it is reduced. Even if the energy of the laser beam L1 is increased in order to maintain the brightness of the illumination field, the energy density of the laser beam L4 at the pupil position P is prevented from becoming excessively high, and is caused by the heat of the immersion objective lens 3. There is an advantage that damage can be prevented.

  Further, according to the microscope illumination device 4 and the microscope 1 according to the present embodiment, the back focal position of the relay lens 9 is obtained by continuously moving the axicon lens 8 in the optical axis direction in the fourth step S4. The pattern diameter of the laser beam L4 collected at Q can be continuously changed. As a result, as shown in FIG. 3, the tilt angle of the laser beam L5 emitted from the immersion objective lens 3 toward the specimen S can be continuously changed. By changing the tilt angle, it is possible to continuously adjust the amount of evanescent light that oozes out to the specimen S side.

In this case, according to the microscope illumination device 4 according to the present embodiment, since the axicon lens 8 does not have power, the condensing position of the laser beam L4 that is transmitted even if it is moved in the optical axis direction is set to the optical axis. There is an advantage that it is not necessary to change the direction, and the NA of the laser beam L4 is not changed.
In other words, since the condensing position is not changed, the laser beam L4 is accurately applied to the pupil position P of the immersion objective lens 3 even if the axicon lens 8 is moved in the optical axis direction in order to adjust the amount of evanescent light oozing out. The Koehler illumination can be sustained by continuing to concentrate well. Further, since the NA is not changed, there is an advantage that the width and brightness of the illumination field can be maintained even if the amount of evanescent light exuded is adjusted.

  In the present embodiment, as an optical element that deflects the laser beam L3 that has passed through the diffraction grating 7, an axicon lens 8 that deflects when transmitting the laser beam L3 is used, as shown in FIG. 5A. Although illustrated, it is not limited to this, and as shown in FIG. 5 (b), the laser beam is deflected by reflection by combining the reflecting surface 13a having the tapered surface and the reflecting surface 14a having the tapered inner surface. Mirrors 13 and 14 may be employed.

  Further, in the present embodiment, in the diffraction grating 7, the laser light L2 is converted into the laser light L3 having the main optical axis on the gradually expanding conical surface and having the NA, but instead of this, FIG. As shown in FIG. 4, a condensing lens 15 for providing NA to the laser light L2 converted into substantially parallel light by the collimating lens 6 is provided, and the diffraction grating 16 is a laser having NA emitted from the condensing lens 15. You may employ | adopt what does not have the power for converting light into the light which has the main optical axis along a conical surface. In this way, the function of the diffraction grating 16 can be simplified and configured easily. Further, a lens that allows parallel light to enter the diffraction grating 16 and imparts NA to the laser light emitted from the diffraction grating 16 may be provided in the subsequent stage of the diffraction grating 16.

  In the present embodiment, the microscope illuminating device 4 that performs total reflection illumination that totally reflects the laser light L5 at the interface between the liquid B and the cover glass G is exemplified. The laser light L5 that is obliquely illuminated from the inclined direction is transmitted without being totally reflected, and is applied to a microscope illumination device that performs dark field illumination for observing fluorescence at a position on the optical axis where the transmitted light does not enter. It may be.

  In the present embodiment, the laser speckle is positioned at a position Q optically conjugate with the pupil position P of the immersion objective lens 3, which is the condensing position of the laser light L4 emitted from the axicon lens 8 of FIG. A reducer (random phase modulation element: not shown) may be arranged to reduce speckle noise included in the laser light irradiated on the specimen S.

1 Microscope 2 Stage 3 Immersion Objective Lens (Condenser Lens)
4 Microscope illumination device 7,16 Diffraction grating (light conversion part)
8 Axicon lenses (optical elements)
15 condenser lens
P pupil position Q conjugate position S specimen S1 first step S2 second step S3 third step S4 fourth step

Claims (7)

The laser beam emitted from the light source is incident, has progressed Yu vital the incident optical axis has and NA the main optical axis distributed on a conical surface extending at an angle to the incident optical axis A light conversion unit for converting into laser light including the emitted light and emitting the laser light;
An optical element having no power that deflects laser light emitted from the light conversion unit in a direction parallel to the incident optical axis;
A microscope illuminator that condenses laser light emitted from the optical element at a pupil position of a condenser lens for illuminating a specimen or a conjugate position thereof.   The microscope illumination apparatus according to claim 1, wherein the light conversion unit is a diffraction grating that deflects laser light and applies NA.   The microscope illumination apparatus according to claim 1, wherein the light conversion unit includes a lens having a positive power that gives NA to laser light, and a diffraction grating that deflects the laser light.   The microscope illumination apparatus according to any one of claims 1 to 3, wherein the light conversion unit converts the laser light into a laser beam continuously distributed over the entire circumference on the conical surface.   The microscope illumination device according to any one of claims 1 to 4, wherein the optical element is provided so as to be movable along an incident optical axis direction of the laser light. The laser beam emitted from the light source, the light has progressed Yu vital the incident optical axis has and NA the main optical axis distributed on a conical surface extending at an angle to the incident optical axis A first step of converting into a laser beam comprising :
A second step of deflecting the laser beam converted in the first step in a direction parallel to the incident optical axis by an optical element having no power;
A third step of condensing the laser beam deflected in the second step at a pupil position of a condenser lens for illuminating the specimen or a conjugate position thereof;
And a fourth step of moving the optical element along an incident optical axis direction of the laser light. A stage with a specimen,
A condensing lens disposed opposite to the specimen mounted on the stage;
A microscope comprising the microscope illumination device according to any one of claims 1 to 5, wherein laser light is incident on the condenser lens.

Images