JP2009282112A - Confocal microscope - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To achieve miniaturization and reduction of cost of a confocal microscope while maintaining an application range of observation of a sample. <P>SOLUTION: The confocal microscope 1 is equipped with a light source 11, a diffusing plate 12, a collimator lens 13, a polarizing plate 14, a polarization beam splitter 15, an imaging lens 16, a quarter-wave plate 17, an objective lens 18, an optical spatial modulator 20, and a CCD camera 29. The optical spatial modulator 20 includes an LCOS (Liquid Crystal on Silicon) is constituted by holding liquid crystal between a mirror-surface state silicon chip where a plurality of pixels are arranged and a surface glass layer, and has structure where a drive circuit for driving each of the pixels is embedded in the back side of the silicon chip. Only light from the pixel being on-controlled is transmitted, so that a spatial optical pattern is projected to a measuring surface of a sample 19 by the optical spatial modulator 20. Then, reflected light from the measuring surface is directly imaged by the CCD camera 29. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a confocal microscope, and more particularly to a confocal microscope capable of simplifying the configuration and performing high-speed sampling.

  Conventionally, a confocal microscope is used to observe a sample by scanning a convergent light spot on the sample and forming an image by forming an image of return light such as fluorescence or reflected light from the sample. . This confocal microscope is used, for example, for observing the shape of a living cell and observing physiological responses in technical fields such as biological and biotechnological fields, and for example for observing the surface of integrated circuits in technical fields such as semiconductors. It is used. In addition, since the confocal microscope has particularly high resolution on the Z axis (in the depth direction), it has been widely used in other applications.

  As such a confocal microscope, as a typical one, for example, a type employing a Nipkow disk system, a type employing a galvanometer mirror, and a type employing a DMD (Digital Micromirror Device) system are known. The Niipou disc method scans the image of the pinhole generated on the sample by mechanically rotating a disc with a plurality of small holes (pinholes). The method using a galvanometer mirror is a light source The laser beam emitted from the laser beam is narrowed in a dot shape, and the image of this point is shaken by a fan that rotates and vibrates in a fan shape, and laser scanning is performed with a bright spot generated on the sample. In the DMD method, the light reflected by the mirror is scanned on the sample by controlling the tilt of the micromirror.

  A confocal microscope is also known that employs a plurality of these methods to selectively illuminate and observe a sample region. In this confocal microscope, for example, light emitted from a light source is incident on a DMD through a condensing lens and a rotary filter, and only the light irradiated on a micromirror region that is on-controlled by the DMD is a microlens of the second Nipou disk. Is incident on. The light that has passed through the microlens is imaged on the pinhole of the first Niipou disk and is imaged on a selected region of the sample by the objective lens. Reflected light such as fluorescent light emitted from a selected region of the sample is reflected by a dichroic mirror in the opposite direction of the incident optical path and imaged on a two-dimensional photodetector by an imaging lens (for example, patent) Reference 1).

JP 2006-220818 A

  Since the confocal microscope described in Patent Document 1 can selectively illuminate and observe a sample region using DMD while improving the light utilization efficiency, it can be used for a wide range of applications of caged reagents and a plurality of minute regions. It is set as the structure which can observe an intracellular response by performing light cancellation stimulus one by one. However, since the Nipkow disk method and the DMD method are adopted, the configuration of the apparatus becomes complicated, and the optical design is simplified by the telecentric optical system, making it impossible to construct a confocal microscope compactly. There is.

  SUMMARY OF THE INVENTION The present invention solves such a problem, and an object of the present invention is to provide a confocal microscope capable of reducing the size and cost while maintaining the application range of sample observation.

  A confocal microscope according to the present invention is a confocal microscope that scans a sample surface with a laser beam and observes the sample with the light on the focal plane, the light source for irradiating the laser beam, and the laser beam from the light source A spatial light modulator made of LCOS that can be reflected by rotating the polarization direction for each pixel constituting a plurality of beam spots, and a plurality of spot lights reflected by the light spatial modulator and projected onto the sample surface. An image pickup device that reads reflected light from the focal plane, and a control unit that controls the light spatial modulator and the image pickup device are provided.

  According to the above configuration, the spatial light modulator that configures a plurality of beam spots is made of LCOS, so that it is compact while maintaining the application range of sample observation while eliminating the need for complicated device configurations such as conventional Niipou discs and DMDs. And cost reduction.

  Further, in the confocal microscope according to the present invention, it is composed of LCOS that reflects reflected light from a plurality of spot lights reflected by the spatial light modulator and projected onto the sample surface by rotating the polarization direction for each pixel. A pinhole array may be further provided, and the control unit may further control the pinhole array.

  In the confocal microscope according to the present invention, the control means may be configured to change at least one of the position, arrangement, and number of the plurality of beam spots of the spatial light modulator.

  In the confocal microscope according to the present invention, the control means may be configured to change at least one of the position, arrangement, and number of the plurality of pinholes of the pinhole array.

  As described above, according to the present invention, it is possible to provide a confocal microscope capable of reducing the size and cost while maintaining the application range of sample observation.

  Next, an embodiment of a confocal microscope according to the present invention will be described with reference to the drawings.

  FIG. 1 is a configuration diagram illustrating an example of a confocal microscope according to an embodiment of the present invention. 2 and 3 are explanatory diagrams for explaining the operation principle of the confocal microscope.

  As shown in FIG. 1, the confocal microscope 1 includes, for example, a light source 11, a diffusion plate 12, a collimator lens 13, a polarizing plate 14, a polarizing beam splitter 15, an imaging lens 16, and a λ / 4 wavelength plate. 17, an objective lens 18, a spatial light modulator 20, and a CCD camera 29.

  The light source 11 is configured by a laser diode or the like, and outputs laser light that is excitation light irradiated on the sample 19. The diffusion plate 12 and the collimator lens 13 make the laser light from the light source 11 parallel light. The polarizing plate 14 changes the laser light that has passed through the diffusion plate 12 and the collimator lens 13 into linearly polarized light that vibrates in one direction.

  The polarization beam splitter 15 separates the light incident through the polarizing plate 14 by the polarization component, transmits the P polarization component to the spatial light modulator 20 and reflects the S polarization component. The spatial light modulator 20 is composed of, for example, a reflective liquid crystal element (LCOS), and is configured by sandwiching liquid crystal between a mirror-like silicon chip in which a plurality of pixels are arranged and a glass layer on the surface. The driving circuit for driving each pixel is embedded on the back side of the silicon chip.

  The spatial light modulator 20 is connected to a control means (not shown) such as a computer, and rotates the polarization direction of the S-polarized component light reflected by the polarization beam splitter 15 for each pixel during on-control. It has a function that can be reflected. The rotation angle amount (phase modulation amount) of each polarization in the spatial light modulator 20 is individually controlled for each pixel by the control means.

  Accordingly, a plurality of beam spots 20b can be generated on the surface 20a of the spatial light modulator 20, and a plurality of spot lights having a scanning pattern in the plane direction (horizontal / vertical direction) of the point array can be generated. Note that at least one of the position, arrangement, and number of the plurality of beam spots 20b can be changed by the control of the control means. Thus, in the confocal microscope 1 of this example, since a plurality of beam spots 20b can be generated by the spatial light modulator 20 made of LCOS, compared with the conventional Nipow disk type or DMD type confocal microscopes, There is no error based on mechanical elements for rotating the Nipkow disk and elements for controlling the arrangement of the micromirror angle unique to the DMD, and the scanning accuracy can be improved.

  The reflected light constituting the plurality of spot lights generated by the spatial light modulator 20 is transmitted through the polarization beam splitter 15 and rotated in the polarization direction by 90 degrees via the imaging lens 16 and the λ / 4 wavelength plate 17. The image is formed on the measurement surface of the sample 19 by the objective lens 18.

  Then, the return light from the measurement surface of the sample 19 passes through the objective lens 18, the λ / 4 wavelength plate 17, and the imaging lens 16 again, and the polarization direction is rotated by 90 degrees. And is focused on the imaging surface of the CCD camera 29.

  According to the confocal microscope 1 having such a configuration, only the light from the pixel that is on-controlled is transmitted by the spatial light modulator 20, and a plurality of spot lights can be projected onto the measurement surface of the sample 19, The reflected light from the measurement surface can be directly imaged by the CCD camera 29. Further, by changing the position of the plurality of beam spots 20b by changing the pixels to be turned on by the spatial light modulator 20, the spot light can be projected onto all the pixels on the entire measurement surface. For this reason, the confocal microscope 1 can be compactly configured without using conventional components such as the Nipkow disk and DMD, and the confocal image can be obtained while reducing the cost.

  That is, for example, in a confocal microscope employing a conventional DMD method, if each DMD micromirror is tilted by ± 10 ° between on and off, the light reflected by the off micromirror is 40 ° to the optical axis. Since reflection is performed at an angle, the center of the CCD element, DMD, and objective lens is arranged on the optical axis in a compact manner so that the light reflected from the off micromirror does not interfere with the light of the on micromirror. It is difficult. In addition, the configuration of the double-sided telecentric optical system is not easy.

  On the other hand, in the confocal microscope 1 of this example, the light reflected by the off pixel remains the S-polarized component and is reflected again by the polarization beam splitter 15, so that it does not interfere with the light of the pixel being on-controlled. The structure can be realized, the telecentric optical system can be easily constructed, and the optical design can be simplified.

  Further, for example, the pixel size in a square DMD is generally about 15 μm, but in the confocal microscope 1 of this example employing the spatial light modulator 20 made of LCOS, the pixel size can be further reduced. . For this reason, the confocal microscope 1 can be configured to be very compact as compared with the Nifo Disc type and DMD type confocal microscopes, and the cost can be reduced.

  Here, as shown in FIG. 2, the operating principle of the confocal microscope is that the light from the light source 41 is focused through the condenser lens 42, transmitted through the pinhole 43 and the beam splitter 44, and passed through the objective lens 45. 49, the reflected light is reflected by the beam splitter 44 through the objective lens 45, and formed on the CCD camera 47 through the pinhole 46 to obtain an image.

  When the entire surface of the sample 49 measured according to such a principle is scanned in the plane direction, as shown in FIGS. 2B and 3A, the shape of the beam spot 31a is in the focused state. As shown in FIG. 3B, the light intensity after the pinhole is small. On the other hand, as shown in FIGS. 2 (a), 2 (c) and 3 (a), the beam spot 31b has a large shape when not in focus, and a pinhole as shown in FIG. 3 (b). Later light intensity will be low. That is, most of the reflected light when not in focus is cut by a pinhole, and information on only the focus position can be obtained by the CCD camera 47.

(How to use light intensity and brightness, maximum brightness)
By utilizing this fact, the entire surface of the sample 49 is scanned in the plane direction while the distance between the sample 49 and the objective lens 45 is changed and scanning on the Z-axis is performed from one focal plane to the other. Thus, for each pixel, the position information on the Z axis where the maximum luminance is obtained can be stored, and the focal position of each pixel can be analyzed to derive the height position of the sample 49. In the current mainstream confocal microscope, in order to increase the image acquisition speed, the entire surface of the sample 49 is scanned with a plurality of point light sources instead of a single point light source, and images are acquired at high speed and with high resolution. Can do.

  FIG. 4 is an explanatory diagram for explaining a spatial light pattern by the optical spatial modulator 20 of the confocal microscope 1 of this example. The plurality of beam spots 20b of the spatial light modulator 20 are constructed as an example of a surface direction scanning pattern of a point array, for example. As shown in FIG. 4, the beam spot of the spatial light modulator 20 is overlapped with the pixel of the CCD camera 47 even when the beam spot formed by the adjacent spatial light modulator 20 pixel is not focused. All pixels can be turned on by simultaneously turning on a plurality of pixels and changing the on-controlled pixels sequentially (for example, a → p in the figure). 49 can scan the entire surface in the plane direction).

  In the example shown in FIG. 4, for example, by changing the on-control 16 times in the spatial light modulator 20, all the pixels can be turned on. In this case, it is possible to obtain 16 images by the CCD camera 47 and obtain the in-focus state from the spot size and brightness of each image to derive the relationship between the height of the sample 49. It becomes.

  As described above, according to the confocal microscope 1 according to the present embodiment, various samples can be observed reliably and accurately. Therefore, the application range of observation compared with conventional confocal microscopes of the Niipou disk method and DMD method It is possible to adopt a compact configuration while maintaining the above (that is, having high versatility regarding observation).

  FIG. 5 is an explanatory diagram for explaining an optical spatial modulator of the confocal microscope. As shown in FIG. 5A, the spatial light modulator 20 of the confocal microscope 1 includes a plurality of pixels 21 when viewed from the surface 20a, and changes the phase of incident light for each pixel 21. As shown in FIG. 4B, the control means is connected to a board on which a control means is mounted via a flexible printed board 22 and a connector 23.

  The spatial light modulator 20 is used together with a polarizer such as a polarizing beam splitter 15 disposed in the optical path of incident light and reflected light, as shown in FIG. An arbitrary spatial light pattern is generated by reflecting the reflected light through the polarizing beam splitter 15 at the inner pixel 21a and reflecting the reflected light at the off-controlled pixel 21b. Can do.

  FIG. 6 is a configuration diagram illustrating an example of a confocal microscope according to another embodiment of the present invention. In the following, portions that overlap with the portions already described are denoted by the same reference numerals and description thereof is omitted. As shown in FIG. 6, the confocal microscope 1 </ b> A includes a pinhole array 20 </ b> B for removing light when the CCD camera 29 is not in focus with the above-described confocal microscope 1. It is different in that the configuration is suitable for acquiring the confocal image in real time.

  The pinhole array 20B is made of LCOS like the above-described spatial light modulator 20, and is configured by sandwiching liquid crystal between a mirror-like silicon chip on which a plurality of pixels are arranged and the glass layer of the surface 20Ba. A drive circuit for driving each pixel 20Bb is embedded on the back side of the silicon chip, and is connected to a control unit (not shown). In synchronization with the spatial light modulator 20, the pixel during the on-control of the spatial light modulator 20 The pixel at the position corresponding to is controlled to be turned on.

  During the ON control, this pinhole array 20B rotates the polarization direction of the reflected light of the P-polarized component from the sample 19 that has passed through the polarizing beam splitter 27 to turn it into S-polarized light, and reflects the polarized beam splitter 27. An image is formed on the CCD camera 29 via the imaging lens 28. The light reflected by the off-pixel remains as the P-polarized component and is transmitted again by the polarization beam splitter 15 and therefore does not enter the CCD camera 29. Therefore, since the shape of the beam spot is small when focused on the surface of the pinhole array 20B, even if the pixel size of the LCOS forming the pinhole array 20B is small, all of the beam spots are high in the CCD camera 29. An image can be formed with light intensity. On the other hand, when the beam spot is not focused, the beam spot has a large shape, so that light is also applied to the pixel under LCOS on-control and the adjacent off-pixel, but only the center portion of the beam spot is affected by the pixel under on-control. Is imaged on the CCD camera 29, the light intensity is low. That is, most of the reflected light when not in focus is cut by the pinhole array 20B, and only the in-focus position information is obtained by the CCD camera 29.

  FIG. 7 shows an image picked up by a CCD camera when a planar sample is tilted and measured with a confocal microscope by the above-described point array surface direction scanning pattern in FIG. ) Represents an image captured by the confocal microscope 1 according to the embodiment of the present invention shown in FIG. 1, and FIG. 7C is a confocal microscope 1A according to another embodiment of the present invention shown in FIG. It represents an image captured by. In the image shown in FIG. 7C, crosstalk to adjacent pixels does not occur. Therefore, in the example shown in FIG. 4, all the pixels are changed by changing the on-control 16 times in the spatial light modulator 20. 2 is obtained, and 16 images are obtained by the CCD camera 47 shown in FIG. 2 to derive the relationship of the height of the sample 49. It is also possible to change the on-control 16 times within the time of image acquisition and to control all pixels to be acquired as one image. In this case, the first control is performed. Represents. Further, for example, by changing the ON control four times in the spatial light modulator 20, all the pixels are turned on, and the height relationship of the sample 49 is obtained by acquiring four images by the CCD camera 47. Can be derived. Alternatively, when the pixel in the embodiment of the present invention shown in FIG. 1 is illustrated as an example, the light spatial modulator 20 performs four on-controls within the time for acquiring one image of the CCD camera 29. By changing and turning on all the pixels, one image acquisition is sufficient to scan the entire surface of the sample 19 and derive the height relationship of the sample 19. As a result, the image of the sample 19 can be accurately formed on the CCD camera 29 in real time.

  As described above, in the confocal microscopes 1 and 1A according to the embodiment of the present invention, the spatial light modulator 20 and the pin made of LCOS that can be produced using the existing semiconductor manufacturing infrastructure instead of the Nipkow disk and DMD. Since the hole array 20B can be used, the confocal microscope can be configured compactly to reduce the cost.

It is a block diagram which shows the example of the confocal microscope which concerns on one Embodiment of this invention. It is explanatory drawing for demonstrating the operation | movement principle of the confocal microscope. It is explanatory drawing for demonstrating the operation | movement principle of the confocal microscope. It is explanatory drawing for demonstrating the spatial light pattern by an optical spatial modulator. It is explanatory drawing for demonstrating the optical spatial modulator of the same confocal microscope. It is a block diagram which shows the example of the confocal microscope which concerns on other embodiment of this invention. It is explanatory drawing for demonstrating the image imaged with the CCD camera of the confocal microscope which concerns on one Embodiment and other embodiment of this invention.

Explanation of symbols

  DESCRIPTION OF SYMBOLS 1,1A ... Confocal microscope, 11 ... Light source, 12 ... Diffuser plate, 13 ... Collimator lens, 14 ... Polarizing plate, 15, 27 ... Polarizing beam splitter, 16, 28 ... Imaging lens, 17 ... λ / 4 wavelength plate 18 ... objective lens, 19 ... sample, 20 ... light spatial modulator, 20B ... pinhole array.

Claims (4)

A confocal microscope that scans a sample surface with a laser beam and observes the sample with the light of the focal plane,
A light source for irradiating the laser beam;
A spatial light modulator composed of LCOS capable of reflecting the laser light from the light source by rotating the polarization direction for each pixel constituting a plurality of beam spots;
An image sensor that reads reflected light from the focal plane from a plurality of spot lights reflected by the spatial light modulator and projected onto the sample surface;
A confocal microscope, comprising: the spatial light modulator and a control unit that controls the imaging device. A pinhole array made of LCOS that reflects the reflected light from the plurality of spot lights reflected by the light spatial modulator and projected onto the sample surface by rotating the polarization direction for each pixel;
The confocal microscope according to claim 1, wherein the control unit further controls the pinhole array.   The confocal microscope according to claim 1, wherein the control unit changes at least one of a position, an arrangement, and a number of the plurality of beam spots of the spatial light modulator.   The confocal microscope according to claim 2, wherein the control unit changes at least one of a position, an arrangement, and a number of the plurality of pinholes in the pinhole array.

Images