JP2014044254A - Microscope illumination optical system, and microscope using the same - Google Patents

Abstract

A microscope illumination optical system capable of forming a dense lattice-shaped illumination light intensity distribution on an object surface while having a compact configuration with a small numerical aperture.
A microscope illumination optical system illuminates an object plane in a microscope that observes a sample placed on the object plane. In the illumination optical system, a plurality of coherent light source regions are arranged apart from each other on the pupil plane 203, and among the distances between the centers of the plurality of light source regions and the pupil center of the illumination optical system, At least one distance is different from the other distances. The illumination optical system forms a second illumination light intensity distribution shifted by half of the grating period with respect to the lattice-shaped first illumination light intensity distribution formed on the pupil plane side of the object plane, and the second illumination light intensity distribution is formed on the object plane. The configuration 206 forms a third illumination light intensity distribution obtained by superimposing the first and second illumination light intensity distributions.
[Selection] Figure 7

Description

  The present invention relates to a microscope illumination optical system that illuminates a sample in a microscope, and more particularly to a microscope illumination optical system suitable for a three-dimensional fluorescence microscope.

  Observation of biological samples with a microscope, in particular a fluorescence microscope, is essential for biological research including medical applications. However, when a thick sample is observed with a normal fluorescence microscope, an image in which images at all height positions where light inside the sample is transmitted is superimposed is observed. That is, in addition to the image of the plane at the height position where the focus is in focus (focusing plane), the blur image of the plane at the height position where the focus is out of focus (non-focusing plane) is superimposed and observed. As described above, in an ordinary fluorescent microscope, it is not possible to selectively separate only an image on a desired focal plane. The effect of selectively separating and extracting only the image of the desired in-focus plane is called a “sectioning effect”.

  A fluorescence microscope configured to obtain a sectioning effect based on various mechanisms is called a three-dimensional fluorescence microscope and is distinguished from a normal fluorescence microscope. When there is a sectioning effect, it is possible to create a three-dimensional three-dimensional image by laminating images of arbitrary focal planes on a computer. That is, anyone can perform the stereoscopic view of the cell arrangement that has been performed in the brain by an experienced pathologist or the like by digital processing.

  As a typical three-dimensional fluorescence microscope, there is a confocal microscope. This is a system that blocks only light coming from the desired in-focus plane and shields light with a low degree of convergence coming from the out-of-focus plane by placing a pinhole at the condensing point of the light coming from the in-focus plane. It is. This method has a high sectioning effect, but since the area that can be imaged at one time is narrow in a dot shape, scanning is necessary to observe the entire area of the sample.

  On the other hand, there is a structured illumination method as a method of realizing a sectioning effect by using image processing by a computer (see Non-Patent Document 1). In this method, a situation is created in which the illumination intensity changes on, for example, a sine wave on the object plane, and a plurality of images in which the sine wave structure is translated are acquired by shifting the phase. Thereafter, the sectioning effect is obtained by processing the plurality of images on the computer. This scheme requires the creation of a sinusoidal structure with controlled phase, ie position, with high accuracy.

  Furthermore, a method of using randomly generated speckle as illumination is also known (see Patent Document 1 and Non-Patent Documents 2 to 6). This method also uses image processing by a computer. However, since the illumination intensity on the object surface depends on random speckles, there is a disadvantage that uneven intensity unevenness is unavoidable in the final image.

US Patent Publication No. 2010/0224796

M. A. A. Neil and T. Wilson, “Method of obtaining optical sectioning by using structured light in a conventional microscope,” Opt. Lett. 22, 1905 (1997). C. Ventalon and J. Mertz, “Quasi-confocal fluorescence sectioning with dynamic speckle illumination,” Opt. Lett. 30, 3350-3352 (2005). C. Ventalon and J. Mertz, “Dynamic speckle illumination microscopy with translated versus randomized speckle patterns,” Opt. Express 14, 7198-7209 (2006). C. Ventalon, R. Heintzmann, and J. Mertz, “Dynamic speckle illumination microscopy with wavelet prefiltering,” Opt. Lett. 32, 1417-1419 (2007). Daryl Lim, Kengyeh K. Chu, and Jerome Mertz, “Wide-field fluorescence sectioning with hybrid speckle and uniform-illumination microscopy,” Opt. Lett. 33, 1819-1821 (2008). Daryl Lim, N. Ford, Kengyeh K. Chu, and Jerome Mertz, “Optically sectioned in vivo imaging with speckle illumination HiLo microscopy” Journal of Biomedical Optics. 16, 016014 (2011).

  Therefore, the present inventor has arranged a plurality of coherent light sources asymmetrically on the pupil plane as a microscope illumination optical system having a high-quality sectioning effect while not requiring high precision in the object plane scanning and illumination system. Proposed illumination optical system. In other words, a plurality of coherent light source regions are arranged apart from each other on the pupil plane, and at least one of the distances between the center of each of the plurality of light source regions and the center of the pupil of the illumination optical system is set to the other distance. We have proposed an illumination optical system with different distances. According to this illumination optical system, the object plane is illuminated with a grid-like illumination light intensity distribution.

  According to the description of Non-Patent Documents 5 and 6, it is desirable that the grid-like illumination interval (pitch) is narrower because the resolution in the horizontal direction can be increased, and this is because between the light source regions on the pupil plane. This is equivalent to increasing the distance.

  However, in reality, since the size (radius) of the pupil has a finite size determined by the numerical aperture of the illumination optical system, the distance between the light source regions also has an upper limit. Increasing the numerical aperture increases the size of the illumination optical system, making it difficult to realize a compact illumination optical system.

  The present invention provides a microscope illumination optical system capable of forming a dense lattice-like illumination light intensity distribution on the object surface and realizing a high-resolution microscope while having a compact configuration with a small numerical aperture. A system and a microscope using the system are provided.

  A microscope illumination optical system according to one aspect of the present invention illuminates an object surface in a microscope that observes a sample placed on the object surface. In the illumination optical system, a plurality of coherent light source regions are arranged apart from each other on the pupil plane, and at least of the distances between the centers of the plurality of light source regions and the pupil center of the illumination optical system. One distance is different from the other. Then, the illumination optical system forms a second illumination light intensity distribution that is shifted by half of the grating period with respect to the grating-like first illumination light intensity distribution formed on the pupil plane side with respect to the object plane, A third illumination light intensity distribution is formed by superimposing the first and second illumination light intensity distributions on the surface.

  Note that a microscope having the microscope illumination optical system and an objective optical system for observing the sample illuminated by the microscope illumination optical system also constitutes another aspect of the present invention.

ADVANTAGE OF THE INVENTION According to this invention, although it is a compact structure with a small numerical aperture, the illumination optical system for microscopes which can form a grid-like illumination light intensity distribution with a high density on an object surface is realizable. By using this illumination optical system for a microscope, a microscope having a high resolution can be realized.

The figure which shows the pupil function P in the premise technique of this invention, and the illumination light intensity distribution in the plane of z = 0 micrometer in a sample when this P is used. A pupil function obtained when the pupil function shown in FIG. 1 is reduced and projected onto a pupil having a numerical aperture that is half the size of the pupil shown in FIG. 1, and illumination on a plane of z = 0 μm in the sample when the pupil function is used. The figure which shows light intensity distribution. The figure showing a line and space. The figure which simulated the intensity distribution in the cross section of y = 0 micrometer when the pattern of FIG. 3 was imaged using the illumination shown by FIG. The figure which simulated the intensity distribution in the cross section of y = 0 micrometer when the pattern of FIG. 3 was imaged using the illumination shown by FIG. The figure which shows typically the effect | action of a birefringent crystal. The figure which shows the example of application with respect to the transmission type fluorescence microscope of the illumination optical system which is an Example of this invention. The figure which shows the illumination light intensity distribution in the plane of z = 0 micrometer in a sample formed of the pupil function in Example 1 of this invention, and the illumination optical system with the pupil function. The figure which represents typically the shape change of the grid-like illumination produced by the effect | action of a birefringent crystal. The figure showing the illumination light intensity distribution which added the effect | action of the birefringent crystal to the illumination light intensity distribution shown by FIG.8 (b). The figure which simulated the intensity distribution in the cross section of y = 0 micrometer in a sample when the pattern of FIG. 3 was imaged by the illumination optical intensity distribution shown by FIG.8 (b). The figure which simulated the intensity distribution in the cross section of y = 0 micrometer in a sample when the effect | action of a birefringent crystal is added to the illumination optical intensity distribution shown by FIG.8 (b), and the pattern of FIG. 3 is imaged. The figure explaining the pupil function of an Example.

  Embodiments of the present invention will be described below with reference to the drawings.

  The illumination optical system for a microscope that is an embodiment of the present invention can be applied to, for example, a three-dimensional microscope that observes a sample as a self-luminous body whose light emission mechanism is fluorescence or phosphorescence. As the type of the microscope, either an episcopic microscope or a transmission microscope may be used.

  As a specific example, the illumination optical system for a microscope according to the embodiment can be applied to a microscope for observing a fluorescently stained sample that is a test sample used in a digital slide scanner. A digital slide scanner is a device that scans a preparation used in biology or pathological examination at high speed and converts it into high-resolution digital data. Furthermore, the illumination optical system for a microscope according to the embodiment can be used as a means for imparting a sectioning effect to, for example, a digital slide scanner including a projection optical system having a large numerical aperture (NA) or a normal fluorescence microscope.

First, an illumination optical system for a microscope proposed by the present inventor, which is a prerequisite technology of the present invention, will be described. In this illumination optical system, P (f, g) shown in FIG. 1A is used as a pupil function (amplitude distribution) P (f, g) on the pupil plane of the illumination optical system. P (f, g) is a triangle formed by three points in the pupil shown by the following coordinate expression (1) in the orthogonal coordinate system on the pupil plane normalized by the radius 1 of the illumination optical system or the three points. A point light source coherent to each other is arranged at three vertices of a triangle similar to the above.
(-1 / √2 + a, 1 / √2 + b)
(-1 / √2 + a, -1 / √2 + b)
(1 / √2 + b, −1 / √2 + a). . . (1)
A and b in these coordinates are real numbers.

  In other words, in the pupil function P (f, g), as shown in FIG. 13, a plurality of light source regions A, B, and C that are coherent with each other are arranged on the pupil plane of the illumination optical system. At least one of the distances dA, dB, dC between the centers cA, cB, cC of the light source regions A, B, C and the center cP of the pupil of the illumination optical system is another distance. And different. The light source region here includes a point light source that can be regarded as having a minute region. The light source region is preferably a region having a size ratio of less than 0.3 with respect to the radius of the pupil. That at least one distance is different from other distances means that all distances may be different, or that only one distance may be different from two or more other same distances. Such an asymmetrical arrangement of the plurality of coherent light sources with respect to the origin (pupil center) is intentionally made in order to obtain resolution in the z direction in a three-dimensional microscope.

  FIG. 1B shows a grid-like illumination light intensity distribution on the object plane that can be actually obtained using the pupil function (amplitude distribution) P (f, g).

  As described above, it is desirable that the lattice interval (pitch) in such lattice illumination is as narrow as possible. However, in reality, it is conceivable that a large illumination optical system having a large numerical aperture cannot be prepared due to restrictions on mechanical arrangement space. Even in such a case, it is necessary not to reduce the resolution in the horizontal direction.

  FIG. 2 (a) shows the point light source arrangement when the point light source arrangement shown in FIG. 1 (a) is reduced into a pupil with half the numerical aperture. The outer circle indicates a pupil range normalized to a radius of 1, and the inner circle indicates a pupil range reduced to half its size (diameter). FIG. 2B shows a grid-like illumination light intensity distribution on the object plane corresponding to the point light source arrangement of FIG. When FIG. 2 (b) is compared with FIG. 1 (b), each bright spot is clearly larger and its period is doubled.

  In order to evaluate how much the resolution in the horizontal direction changes when the pitch of the grid illumination changes in this way, consider a fluorescent object having a line and space pattern as shown in FIG. The line width or half pitch is 1 μm.

  Contrast when imaging the pattern shown in FIG. 3 using the illumination shown in FIG. 1B and the illumination shown in FIG. 2B (in this case, the intensity when viewed in a cross section of y = 0 μm) Distribution). 4 shows the contrast when the illumination shown in FIG. 1B is used, and FIG. 5 shows the contrast when the illumination shown in FIG. 2B is used. The difference in contrast between the two is clear, and FIG. 4 is overwhelmingly higher in contrast and has higher horizontal resolution. Therefore, it is better to use the illumination of FIG. 1B where the pitch of the grid illumination is narrower than the illumination of FIG.

  However, in practice, it is often impossible to realize an illumination optical system having a large pupil as typified by FIG.

  Therefore, in this embodiment, as shown in FIG. 2A, a plurality of coherent light sources are arranged in a pupil having a small diameter, and a high density lattice-like illumination is performed using a simple configuration such as a birefringent crystal. Form on the object surface. When a birefringent crystal is used, the extraordinary ray generated by the birefringence phenomenon of the birefringent crystal is shifted by half the lattice period with respect to the lattice-like illumination (first illumination light intensity distribution) formed by the original normal ray. A grid-like illumination (second illumination light intensity distribution) is formed separately. Then, by superimposing the lattice-like illumination formed by the normal ray and the extraordinary ray while the lattice periods are shifted from each other by a half cycle, a higher-density double lattice-like illumination (third illumination light intensity distribution: , Also referred to as a double-grid illumination light intensity distribution) on the object plane.

  A birefringent material typified by calcite or the like separates the light path into two according to the polarization direction of the light. This is shown in FIG. If the plane perpendicular to the paper surface is the cleavage plane of the birefringent crystal, the light oscillating parallel to the paper surface and the light oscillating perpendicularly to the surface become two normal rays and extraordinary rays as shown in the figure. After passing through the crystal plate, it proceeds again in the direction perpendicular to the crystal plate. In order to make both the intensities of light uniform, for example, oblique linearly polarized light having both components in a direction parallel to the paper surface and a direction perpendicular to the paper surface with equal intensity may be prepared. Further, the thickness of the crystal flat plate is set so that the amount of shift of the lattice illumination is half the lattice period of the lattice illumination. Such a crystal flat plate is arranged on the pupil plane side of the object plane on which the sample is arranged.

  Further, as a configuration other than the configuration using the birefringent crystal as described above, the first and second illumination lights described above are configured by using a pair of point light sources on the pupil plane as a pair of two incoherent point light sources. You may implement | achieve 3rd illumination light intensity distribution which formed intensity distribution and overlapped these. Furthermore, the exposure is performed twice with a time difference, and the illumination light intensity distribution (first and second illumination light intensity distributions) for each time is laterally shifted by half of the grating period of the grating-like illumination ( That is, a configuration that realizes the third illumination light intensity distribution) may be employed.

  As described above, according to the illumination optical system for a microscope according to the present embodiment, an image having high horizontal resolution by a compact illumination optical system when used in combination with the method described in Non-Patent Documents 5 and 66. Can be obtained.

  Next, a preferred arrangement example of the illumination optical system of the present embodiment in the three-dimensional fluorescence microscope will be described with reference to FIG. Here, a case where a birefringent crystal is used as a configuration for forming a double grating-like illumination light intensity distribution will be described. In FIG. 7, reference numeral 100 denotes a transmission type three-dimensional fluorescence microscope.

  Reference numeral 200 denotes an illumination optical system, which has a configuration that can be added to a microscope main body constituted by the objective lens 102 and the image sensor 103. Reference numeral 207 denotes an object (sample) arranged on the object plane.

  In the illumination optical system 200, 201 is a coherent light source, and a laser having a wavelength capable of exciting a fluorescent sample can be used. Reference numeral 202 denotes an optical element such as a diffraction grating, a prism, or an optical fiber, which has an action of dividing one beam emitted from the light source 201 into a plurality of (for example, three) beams. The optical element 202 is not limited to the diffraction grating, the prism, or the like, and any optical element can be used as long as it can realize the pupil function shape characterized by the present embodiment on the pupil plane 203 of the illumination optical system 200. The pupil function shape forming method in the present embodiment can be realized by a method that is easy for an engineer related to a microscope or a semiconductor exposure apparatus, and for example, a computer-generated hologram (CGH) can be used.

  The divided beam passes through the lens 205 and the birefringent crystal 206 via the mirror 204, and illuminates the object 207 with a double grating-like illumination light intensity distribution. The fluorescence emitted from the object 207 passes through the objective lens (objective optical system) 102 and forms an image on the image sensor 103. An image obtained by imaging by the image sensor 103 and displayed on a monitor (not shown) is observed by an observer.

  As described above, the illumination optical system of the present embodiment can illuminate the object plane with a double grating-like illumination light intensity distribution having a narrow grating interval with a simple configuration. Moreover, the illumination optical system of the present embodiment can be added (retrofitted) to an ordinary three-dimensional fluorescence microscope. Further, when the illumination optical system of the present embodiment is used, even if the grid-like illumination light intensity distribution is slightly displaced, it is integrated in a region including the lattice distribution, so that the final image quality is not affected.

  Hereinafter, more specific examples of the illumination optical system for a microscope including the above-described birefringent crystal will be described.

Example 1 is an illumination optical system for a microscope having a configuration as shown in FIG. 7, and has optical parameters of wavelength λ = 500 nm and NA = 0.7. The line and space pattern shown in FIG. 3 is adopted as the object. As shown in FIG. 1A, the pupil function of the illumination optical system has three points in the pupil represented by the following coordinate expression (2) or three vertices of a triangle similar to the triangle formed by the three points. This is expressed as arrangement of coherent point light sources. In the formula (2), a and b are a = 0.2 and b = 0.1. This shows a point light source arrangement that fits within a pupil of about half the radius of FIG. 1 (a) as shown in FIG. 8 (a).
(-1 / 2√2 + a, 1 / 2√2 + b)
(-1 / 2√2 + a, -1 / 2√2 + b)
(1 / 2√2 + b, −1 / 2√2 + a). . . (2)
FIG. 8B shows lattice-like illumination (first illumination light intensity distribution) formed on the object plane by this illumination optical system (before insertion of the birefringent crystal).

  FIG. 9 shows changes in illumination shape before and after the birefringent crystal is inserted at a position closer to the pupil plane than the object plane in the illumination optical system. In the figures showing before and after the insertion of the birefringent crystal, the ellipse blacked out shows a lattice-like illumination (first illumination light intensity distribution) formed by normal rays that have passed through the birefringent crystal. Further, in the figure after the insertion of the birefringent crystal, an ellipse indicated by hatching indicates lattice-like illumination (second illumination light intensity distribution) formed by extraordinary rays generated in the birefringent crystal. A double lattice-like illumination (third illumination light intensity distribution) having a high density, that is, a small lattice interval is formed by overlapping the black-painted ellipse and the hatched ellipse. Since the density of the lattice-shaped illumination is increased by the insertion of the birefringent crystal, the area of the unit cell represented by a square in the drawing is small (about 1 / √2 times).

  Further, FIG. 10 shows a simulation result of double lattice illumination expected to be formed by inserting a birefringent crystal. As can be seen from FIG. 8A, since each illumination point in the grid illumination has an elliptical shape, the illumination light intensity distributions overlap each other in the major axis direction, and as a result, a plurality of oblique illuminations as a whole. looks like.

  The effect of the birefringent crystal will be described with reference to FIGS. FIG. 11 shows a simulation result of the illumination light intensity distribution in the cross section at y = 0 μm when the line and space pattern shown in FIG. 3 is imaged without inserting the birefringent crystal into the illumination optical system. On the other hand, FIG. 12 shows the illumination light intensity distribution in the cross section at y = 0 μm when the pattern of FIG. 3 is imaged by adding the action of the birefringent crystal to the illumination light intensity distribution shown in FIG. Simulation results are shown. When these are compared, it is apparent that the insertion of the birefringent crystal shown in FIG. 12 has a high contrast and excellent uniformity. This is because the interval between the illumination points of the lattice illumination can be reduced by the double lattice illumination by inserting the birefringent crystal.

  When imaging was performed with the number of pixels of 256 × 256 × 256 using a three-dimensional microscope using the illumination optical system of Example 1, the time required for the processing was within one minute on a workstation equipped with a 3.33 GHz CPU. is there. By creating a dedicated program and optimizing and using hardware devices such as a parallel distributed environment and a graphic accelerator, it is possible to obtain an image in a time shorter than the time required for scanning by the confocal microscope.

  Each embodiment described above is only a representative example, and various modifications and changes can be made to each embodiment in carrying out the present invention.

An illumination optical system that can be used in a microscope such as a fluorescent microscope or a digital slide scanner can be provided.

200 Illumination Optical System 201 Coherent Light Source 203 Pupil (Lens) of Illumination Optical System
206 Birefringent crystal

Claims (5)

An illumination optical system for illuminating the object surface in a microscope for observing a sample placed on the object surface,
A plurality of coherent light source regions are arranged apart from each other on the pupil plane of the illumination optical system,
Of the distances between the centers of the plurality of light source regions and the center of the pupil of the illumination optical system, at least one distance is different from the other distances,
The illumination optical system forms a second illumination light intensity distribution that is shifted by half of the grating period with respect to the grating-like first illumination light intensity distribution formed on the pupil plane side with respect to the object plane, An illumination optical system for a microscope having a configuration in which a third illumination light intensity distribution is formed by superimposing the first and second illumination light intensity distributions on an object plane.   2. The illumination optical system for a microscope according to claim 1, wherein the sample is a self-luminous body.   The illumination optical system for a microscope according to claim 2, wherein a light emission mechanism of the sample which is the self-luminous material is fluorescence or phosphorescence.   The illumination optical system for a microscope according to any one of claims 1 to 3, wherein the illumination optical system is used for a transmission microscope or an episcopic microscope. The illumination optical system for a microscope according to any one of claims 1 to 3,
A microscope comprising: an objective optical system for observing a sample disposed on an object surface illuminated by the microscope illumination optical system.