JP2004013117A - Microscopic illuminator - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a microscopic illuminator wherein a convergence spot position formed on an entrance pupil surface of a condenser lens can be freely set to an arbitrary position on the entrance pupil surface. <P>SOLUTION: The microscopic illuminator has a light source 1, at least one scanner 3 which causes a luminous flux 2 from the light source 1 to scan, a condenser lens for converging the scanning light on an object surface, a relay lens 4 for converging the scanning luminous flux on an entrance pupil surface 1 of the condenser lens 5, a driving circuit 10 for driving the scanner 3, a control circuit 11 for giving a control signal for controlling the scanning state of the scanner 3, to the driving circuit 10, and a control condition storage circuit for storing a control condition of the control circuit 11 therein. <P>COPYRIGHT: (C)2004,JPO

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a microscope illuminating device, and more particularly, to a microscope illuminating device capable of freely controlling the position of a condensed spot focused on an entrance pupil plane of an objective lens.
[0002]
[Prior art]
2. Description of the Related Art Conventionally, there has been a total reflection fluorescence microscope in a fluorescence microscope in which a light beam focused on an entrance pupil plane of an objective lens generates an evanescent wave as excitation light and observes fluorescence. In this method, the excitation light is totally reflected at the interface between the glass substrate holding the sample and the sample, and the sample is irradiated with an evanescent wave. The fluorescent image of that part can be observed.
[0003]
Examples of illumination light used for such a total reflection microscope are disclosed in JP-A-9-159922 and JP-A-2001-272606. In these disclosed examples, laser light emitted from a laser light source or an optical fiber is used to form an objective lens via a relay lens for forming a condensed spot on an entrance pupil plane of the objective lens and a beam splitter for changing an optical path of illumination light. Is focused near the outer periphery of the entrance pupil of the light source. And moving the light emitting end face of the light source or the optical fiber in a direction perpendicular to the optical axis so that the laser beam is irradiated to the sample at a critical angle or more at which the laser beam is totally reflected at the interface between the cover glass (or slide glass) and the sample. Alternatively, the position of the beam splitter is moved up and down to achieve the total reflection condition.
[0004]
[Problems to be solved by the invention]
However, in the above disclosed example, the condensed spot on the entrance pupil plane of the objective lens is set to only one point that satisfies the condition of total reflection. There is a problem that an image obtained by the set position has anisotropy.
[0005]
In addition, when observing an image while changing the position of the focused spot, every time the position of the focused spot is changed, it is necessary to adjust the focused spot position so that the total reflection condition is satisfied while viewing the image. However, it is complicated and it takes time to obtain a desired image.
[0006]
For this reason, there is a demand for a microscope illuminating device capable of freely controlling the position of a condensed light spot converged on an entrance pupil plane of an objective lens.
[0007]
The present invention has been made in view of the above-described problems, and provides a microscope illumination device that can freely set a condensing spot position formed on an entrance pupil plane of a condenser lens to an arbitrary position on the entrance pupil plane. It is intended to be.
[0008]
[Means for Solving the Problems]
In order to achieve the above object, according to the present invention, a light source, at least one scanner that scans a light beam from the light source, a condenser lens that condenses the scanned light beam on an object surface, and the condenser lens A relay lens for condensing the scanned light beam on the entrance pupil plane of the scanner, a driving circuit for driving the scanner, a control circuit for providing a control signal to the driving circuit for controlling a scanning state of the scanner, and the control And a control condition storage circuit for storing control conditions of the circuit.
[0009]
In the microscope illumination device of the present invention, it is preferable that the relay lens is disposed at a position where the object-side focal plane of the scanner and the condenser lens is substantially optically conjugate.
[0010]
Further, it is preferable that the microscope illumination device of the present invention includes a light modulation circuit for modulating the light intensity of the light source, and a modulation condition storage circuit for storing a modulation condition of the light modulation circuit.
[0011]
Further, in the microscope illumination device of the present invention, a light modulation member disposed on an optical path between the light source and the scanner, a second modulation circuit for controlling the light modulation member, and a second modulation circuit And a second modulation condition storage circuit for storing the modulation condition.
[0012]
Further, in the microscope illumination device of the present invention, a step of scanning the light beam from the light source with the scanner, a step of detecting light intensity corresponding to the scanning position on the object surface, and the step of detecting the detected light intensity information And selecting the coordinates of a circle passing through the three different positions on the basis of the above, and storing the determined coordinates of the circle in the control condition storage circuit. Is desirable.
[0013]
The present invention also provides a microscope including the microscope illumination device.
[0014]
DETAILED DESCRIPTION OF THE INVENTION
An embodiment of the present invention will be described with reference to the drawings.
[0015]
FIG. 1 shows a schematic configuration diagram of a microscope having a microscope illumination device according to a first embodiment of the present invention, and FIG. 2 shows an example of a scanning pattern of a condensed spot in the first embodiment. FIG. 3 is a schematic configuration diagram of a microscope having a microscope illumination device according to a second embodiment of the present invention, and FIG. 4 is a diagram illustrating an example of a scanning pattern of a condensed spot and a light intensity distribution according to the second embodiment. Show. FIG. 5 is a schematic configuration diagram when the microscope illumination device according to the present invention is used for a total internal reflection microscope.
(1st Embodiment)
In FIGS. 1 and 2, in the microscope illumination device according to the first embodiment, a laser beam 2 emitted from a laser light source 1 enters a two-dimensional galvano scanner 3 and is scanned by the two-dimensional galvano scanner 3. Numeral 2 enters the relay lens 4 and is collected near the entrance pupil plane I of the condenser lens 5. The condensed spot 21 is irradiated on the specimen 6 by the condenser lens 5 to illuminate the specimen 6. The relay lens 4 is disposed at a position where the object-side focal plane of the two-dimensional galvano scanner 3 and the condenser lens 5 is substantially optically conjugate. Further, a control device A including a drive circuit 10 for driving the two-dimensional galvano scanner 3, a control circuit 11 for controlling the drive circuit 10, and a storage device 12 having a control condition storage circuit for storing a programmed control signal is provided. The two-dimensional galvano scanner 3 scans a desired area on the entrance pupil plane I with the laser light 2 based on a control signal from the control device A. The emitted light 7 from the sample 6 is condensed by the objective lens 8, forms an image on the camera 9, and is observed as a sample image on a monitor (not shown) or the like.
[0016]
By using a personal computer or the like for the control device A, the position of the converging spot 21 on the entrance pupil plane I of the condenser lens 5 is accurately determined, and the scanning speed and the scanning area of the converging spot 21 are freely changed. It becomes possible.
[0017]
FIG. 2 shows an example of a scanning pattern of the converging spot 21 formed by the microscope illumination device of the first embodiment. In this example, a case is shown in which the converging spot 21 is controlled to scan in the shape of a ring 22 near the outer periphery on the entrance pupil plane I of the condenser lens 5.
[0018]
In such illumination light, when the condensed spot 21 is rapidly scanned in the shape of the ring 22 in the direction indicated by the arrow in FIG. 2 or in the direction opposite to the direction indicated by the arrow, the exposure time of the camera 9 is converged. If the spot 21 is longer than the time required for one round of the ring 22, the image formed on the camera 9 can be regarded as an image obtained by illuminating the specimen 6 with illumination light having the shape of the ring 22.
[0019]
Further, the microscope illumination device of the first embodiment can be used as an illumination device of a total reflection microscope. In the total reflection microscope, the specimen 6 is illuminated with an evanescent wave. Therefore, in order to make the illumination light incident at a critical angle (total reflection angle), it is necessary to strictly condense the illumination light at a predetermined position on the outer peripheral portion on the entrance pupil surface of the condenser lens 5. The aperture portion that can be used when realizing total reflection is a portion that is approximately 1.35 or more in terms of numerical aperture. For example, when a condenser lens 5 having a numerical aperture of 1.45 is used, an area where total reflection illumination can be performed. Is about 7% or less of the numerical aperture. In practice, it is necessary to form the converging spot 21 in a region of the ring 22 having a width of about 300 microns. At this time, the size of the converging spot 21 is about 25 microns. As described above, in the total internal reflection microscope using the evanescent wave, it is essential to accurately align the converging spot 21 narrowed down to the area of the ring 22 having a width of about 300 μm. In the microscope illuminating device of the embodiment, the position of the condensed spot 21 can be controlled by the two-dimensional galvano scanner 3 based on a signal from the control device. In comparison, it is possible to position the light-converging spot 21 with high accuracy and easily.
[0020]
In addition, since the converging spot 21 can be easily formed in the area shown by the ring 22 in FIG. 2, it is possible to obtain an image without anisotropy.
[0021]
An example of a procedure for determining scanning conditions for scanning the converging spot 21 in the shape of the ring 22 as described above will be described.
[0022]
Let X-Y be the orthogonal coordinate axis passing through the center O of the entrance pupil plane I. A control signal is input from the control device A to the two-dimensional galvano scanner 3 so as to scan the focused spot 21 on the X-axis over the diameter of the entrance pupil plane I, and the camera 9 observes the emitted light 7 at this time. At this time, a water drop substantially equal to the biological characteristics may be used instead of the specimen 6. When the condensed spot 21 scans from the center O on the X axis, the light intensity of the emitted light 7 at the center O becomes the maximum, and the light intensity slightly decreases as the light scans from the center O rightward in the + X direction. The intensity of light emitted near the boundary that becomes the condition sharply decreases and becomes substantially zero. The coordinates when the value becomes substantially zero (or the inflection point when the value changes to substantially zero) are stored in the storage device 12 as X1. Similarly, scanning is performed in the −X direction, and the coordinates X2 at which the light intensity becomes substantially zero (or an inflection point when the light intensity changes to substantially zero) are stored in the storage device 12. In the Y-axis direction, the coordinates Y1 and the coordinates Y2 are stored in the storage device 12 in the same procedure.
[0023]
The coordinates of a circle passing through the four stored coordinates are obtained by calculation, scan coordinate data of the shape of the ring 22 of the condensed spot 21 is created, and stored in the control condition storage circuit of the storage device 12. By controlling the two-dimensional galvano scanner 3 using the data stored in the control condition storage circuit as a control condition, a scanning pattern like the shape of the ring 22 can be formed. In addition, it is possible to create arbitrary scan data without being limited to the shape of the ring 22. Further, the light intensity of the emitted light can be detected by means other than the camera 9, for example, visually. In the above description, the coordinates of a circle are obtained using four coordinates. However, if there are at least three coordinates, the coordinates of a circle can be obtained.
[0024]
The two-dimensional galvano scanner 3 is shown as a member for scanning the laser beam 2 from the laser light source 1, but any other two-dimensional scanner can be used. In addition, a two-dimensional scanner may be configured by combining a one-dimensional scanner, or an acousto-optic device or the like may be used.
(2nd Embodiment)
Next, a second embodiment of the present invention will be described with reference to the drawings.
[0025]
FIG. 3 is a schematic configuration diagram of a microscope having a microscope illumination device according to a second embodiment of the present invention, and FIG. 4 is a diagram illustrating an example of a scanning pattern of a condensed spot and a light intensity distribution according to the second embodiment. Show.
[0026]
The second embodiment differs from the first embodiment in that the second embodiment has an optical modulation circuit for modulating the laser light intensity of the laser light source 1. Members equivalent to those in the first embodiment are denoted by the same reference numerals, and description thereof is omitted.
[0027]
As shown in FIG. 3, the microscope illumination device according to the second embodiment has a laser light source 1 (for example, a semiconductor laser) of a microscope illumination device having substantially the same configuration as the first embodiment. A laser light modulation circuit 31 for modulating the intensity is provided. The laser light modulation circuit 31 modulates the laser light source 1 based on the laser light modulation condition from the modulation condition storage circuit provided in the storage device 12 of the control device A, and emits the modulated laser light 2.
[0028]
In this way, the microscope illumination device according to the second embodiment can modulate the light intensity of the laser beam 2 together with the scanning of the converging spot 21 by the two-dimensional galvano scanner 3.
[0029]
FIG. 4A is an example of a scanning pattern of the converging spot 21, and FIG. 4B is a diagram showing a light intensity distribution corresponding to the scanning pattern of FIG. 4A. The case where the converging spot 21 is scanned over the entire area on the entrance pupil plane I of the condenser lens 5 and the light intensity of the laser beam 2 is increased near the outer periphery of the entrance pupil plane I as compared with the central part of the entrance pupil plane I Is shown. As described above, illumination with different brightness can be performed depending on the position on the entrance pupil plane I, a super-resolution illumination optical system can be realized, and the resolution and the depth of focus of an observation image can be easily improved.
[0030]
Further, by performing control such that the laser beam 2 is turned on at the outer peripheral portion on the entrance pupil plane I and the laser beam is turned off at other portions, the same total reflection illumination as in the first embodiment can be realized. Can be.
[0031]
Further, as shown in FIG. 4 (c), it is possible to scan the entrance pupil plane I so as to have substantially uniform brightness, and it is possible to freely control the manner of scanning.
(Third embodiment)
Next, a third embodiment of the present invention will be described with reference to the drawings.
[0032]
FIG. 5 is a schematic configuration diagram of a microscope having a microscope illumination device according to a third embodiment of the present invention.
[0033]
The third embodiment is different from the second embodiment in that a light modulation member 42 having a second modulation circuit 41 in an optical path between the laser light source 1 and the two-dimensional galvano scanner 3 (for example, an acousto-optical element ( AOM)), a second modulation condition storage circuit is provided in the storage device 12 to store the modulation conditions, and the light intensity of the laser beam 2 is modulated based on the stored modulation conditions. That is. With such a configuration, the same operation and effect as those of the second embodiment can be obtained, and thus detailed description is omitted.
(Fourth embodiment)
Next, a microscope illumination device according to a fourth embodiment of the present invention will be described with reference to the drawings. FIG. 6 shows a microscope illumination device according to the fourth embodiment. In the fourth embodiment, a case is described in which a microscope illumination device is applied to an inverted cubic microscope. Members equivalent to those in the first, second, and third embodiments are denoted by the same reference numerals, and description thereof is omitted.
[0034]
The laser light 2 from the laser light source 1 is scanned by the two-dimensional galvano scanner 3, and the laser light passing through the relay lens 4 is reflected by the beam splitter 51 and condensed on the entrance pupil plane I of the objective lens 8. The sample 6 is irradiated through the objective lens 8. Light (fluorescence) from the sample 6 is condensed by the objective lens 8, passes through the beam splitter 51 and the laser cut filter 52, and is then imaged on the camera 9 by the imaging lens 53 to form an optical image of the sample 6 ( Fluorescent image) is observed.
[0035]
Illumination light for irradiating the sample 6 is applied to the two-dimensional galvanomirror 3 so that a scan of the shape of the ring 22 as shown in FIG. 2 is performed on the entrance pupil plane I of the objective lens 8 based on a control signal from the control device A. Is controlled, so that the sample 6 can be totally reflected. As a result, the specimen 6 emits fluorescence in the vicinity of the interface between the slide glass and the specimen by receiving the evanescent wave, so that a fluorescent image near the interface can be observed.
[0036]
Other operations and effects are the same as those of the first, second, and third embodiments, and thus description thereof is omitted.
[0037]
As described above, according to the present invention, it is possible to easily set the total reflection condition, move the focused spot to an arbitrary position on the entrance pupil plane at an arbitrary speed, and modulate the light intensity. Become.
[0038]
The above-described embodiment is merely an example, and is not limited to the above-described configuration and shape. Modifications and changes can be made as appropriate within the scope of the present invention.
[0039]
【The invention's effect】
As described above, in the present invention, the position of the condensed spot formed on the entrance pupil plane of the condenser lens can be freely set to an arbitrary position on the entrance pupil plane, and the illumination method of the microscope can be diversified.
[Brief description of the drawings]
FIG. 1 is a schematic configuration diagram of a microscope having a microscope illumination device according to a first embodiment of the present invention.
FIG. 2 shows an example of a scanning pattern of a condensed spot in the first embodiment.
FIG. 3 is a schematic configuration diagram of a microscope having a microscope illumination device according to a second embodiment of the present invention.
FIG. 4A shows a scanning pattern of a converging spot in the second embodiment, and FIGS. 4B and 4C show examples of light intensity distribution.
FIG. 5 is a schematic configuration diagram of a microscope having a microscope illumination device according to a third embodiment of the present invention.
FIG. 6 is a schematic configuration diagram of a total internal reflection microscope having a microscope illumination device according to a fourth embodiment of the present invention.
[Explanation of symbols]
REFERENCE SIGNS LIST 1 laser light source 2 laser light 3 two-dimensional galvano scanner 4 relay lens 5 condenser lens 6 sample 7 emission light 8 objective lens 9 camera 10 drive circuit 11 control circuit 12 storage device 21 focusing spot 22 ring 31 laser light modulation circuit 41 second Modulating circuit 42 Light modulating member 51 Beam splitter 52 Laser cut filter 53 Imaging lens A Controller I Entry pupil plane

Claims (6)

A light source,
At least one scanner for scanning a light beam from the light source;
A condenser lens for condensing the scanned light beam on an object surface,
A relay lens for condensing the scanned light beam on the entrance pupil plane of the condenser lens,
A driving circuit for driving the scanner,
A control circuit for providing a control signal for controlling a scanning state of the scanner to the drive circuit;
A control condition storage circuit for storing control conditions of the control circuit;
A microscope lighting device, comprising: 2. The microscope illumination device according to claim 1, wherein the relay lens is arranged at a position where the object-side focal plane of the scanner and the condenser lens is substantially optically conjugate. The microscope illumination device according to claim 1,
A light modulation circuit for modulating the light intensity of the light source,
A modulation condition storage circuit for storing a modulation condition of the light modulation circuit,
A microscope illumination device, comprising: The microscope illumination device according to claim 1,
A light modulation member arranged in an optical path between the light source and the scanner,
A second modulation circuit that controls the light modulation member;
A second modulation condition storage circuit that stores a modulation condition of the second modulation circuit;
A microscope illumination device, comprising: The microscope illumination device according to claim 1,
Scanning the light beam from the light source with the scanner;
Detecting light intensity corresponding to the scanning position on the object surface,
Selecting at least three different positions based on the detected light intensity information and obtaining coordinates of a circle passing through the three different positions;
Storing the determined coordinates of the circle in the control condition storage circuit;
A microscope lighting device, comprising: A microscope comprising the microscope illumination device.

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