JP2013097132A - Imaging device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an imaging device that can increase the focus depth in an imaging optical system and acquire an image with high resolution by irradiating a sample with illumination light in a proper polarization state.SOLUTION: An imaging device 100 includes: an imaging optical system 140 that forms an image of a sample 130 irradiated with illumination light from a light source 111; and an image pickup element 120 that takes an image of the sample 130 of which image has been formed by the imaging optical system 140. The imaging device further includes: a birefringent parallel plate 50 on an optical path between the sample 130 and the image pickup element 120, the plate having birefringence to illumination light; and polarization state switching means 113 that can switch the polarization state of the illumination light to unpolarized light or circularly polarized light, as well as polarized light in a radial direction to the optical axis of the imaging optical system 140 or polarized light in a tangential direction. The direction of the optical axis of the birefringent parallel plate 50 is parallel to the optical axis direction of the imaging optical system 140.

Description

  The present invention relates to an imaging device including an optical member having birefringence.

  When a sample is imaged by an imaging apparatus such as a microscope, the sample or the imaging element may be displaced in the optical axis direction, and the acquired image may be blurred due to defocusing. In general, since the sample and its image are in a conjugate relationship, a shift in the optical axis direction of the imaging optical system of the sample and the image sensor is referred to as defocus without being distinguished. If the defocus is small, the image blur is small and the image can be recognized. However, if the defocus is large, the image blur increases and the image cannot be recognized. Here, the depth of focus is a range in which an image of the sample can be recognized even if the sample or the imaging device is displaced in the optical axis direction. In order to image a fine sample in such an imaging apparatus, it is necessary to increase the numerical aperture (NA) of the imaging optical system. However, when the NA of the imaging optical system increases, the depth of focus decreases.

  Therefore, a bifocal optical system has been proposed as a means for solving the problem of increasing the depth of focus while increasing the NA of the imaging optical system. The bifocal optical system is an optical system that separates the light beam emitted from the sample and forms an image at two focal positions in the optical axis direction, and is an optical member having anisotropy (birefringence). A refractive member (such as crystal or calcite) is used. The light rays that pass through the birefringent member have two different refractive indexes: an ordinary ray whose polarization direction is perpendicular to the optical axis defined for each birefringent member, and an extraordinary ray whose polarization direction is parallel. Are separated into light beams having

  Patent Document 1 proposes an image processing apparatus capable of changing the focal position by switching the polarization state of illumination light by a double-focus optical system using a birefringent crystal. In this image processing apparatus, the sample is imaged at two focal positions, the pixels of the two acquired images are compared with each other, and the one with less blur is selected for all the pixels, and the image is recombined. To increase the depth of focus. Further, Patent Document 2 proposes an endoscope that defines an optimal double focus position in a configuration in which a crystal optical element having birefringence is inserted into an imaging optical system. Further, it is also described that the focus position is changed by switching the illumination light of the imaging optical system between polarized light oscillating perpendicularly and polarized light oscillating parallel to the optical axis of the crystal optical element.

JP-A-11-32251 JP 2007-97652 A

  However, the image processing apparatus in Patent Document 1 requires complicated image processing, and the time required for image acquisition becomes long. In addition, since the resolution of the image is reduced due to the influence of aberrations and refractive index dispersion caused by the birefringent member, when it is not necessary to increase the depth of focus, the illumination light of the imaging optical system is converted into a high-resolution image. It is desirable to obtain a polarization state for acquisition. However, in any of the above-mentioned patent documents, only the polarization state parallel or perpendicular to the optical axis of the birefringent crystal is described, and appropriate polarization for obtaining a high-resolution image is described. There is no description about the state.

  SUMMARY OF THE INVENTION An object of the present invention is to provide an imaging apparatus that can illuminate a sample with illumination light in an appropriate polarization state and can realize an increase in depth of focus or high-resolution image acquisition in an imaging optical system.

  In order to achieve the above object, an imaging apparatus according to an aspect of the present invention includes an imaging optical system that forms an image of a sample illuminated by illumination light from a light source, and the sample imaged by the imaging optical system. A birefringent parallel plate disposed on an optical path between the sample and the image sensor and having birefringence with respect to the illumination light, and the illumination light. Polarization state switching means capable of switching between a non-polarized or circularly polarized light and a radially polarized or tangentially polarized light with respect to the optical axis of the imaging optical system, and the birefringence The direction of the optical axis of the parallel plate is a direction parallel to the optical axis direction of the imaging optical system.

  Further objects and other features of the present invention will be made clear by the preferred embodiments described below with reference to the accompanying drawings.

  ADVANTAGE OF THE INVENTION According to this invention, the imaging device which can illuminate a sample with the illumination light of a suitable polarization state, and can implement | achieve the expansion of the depth of focus in an imaging optical system, or high-resolution image acquisition can be provided.

1 is a schematic diagram of an imaging apparatus according to an embodiment of the present invention. The principal part schematic for demonstrating the optical path of the illumination light which concerns on embodiment of this invention. The figure for demonstrating suitable thickness of the birefringent parallel plate which concerns on embodiment of this invention. The schematic diagram which showed the polarization state of the illumination light which concerns on embodiment of this invention. Schematic of the sample which concerns on embodiment of this invention. The principal part schematic when using the birefringent parallel plate which concerns on embodiment of this invention as a cover glass. The schematic diagram which showed the light intensity distribution of the illumination light which concerns on embodiment of this invention. The figure for demonstrating the effect in Example 1 of this invention. The figure for demonstrating the effect in Example 2 of this invention.

  Hereinafter, preferred embodiments of the present invention will be described with reference to the drawings, but the present invention is not limited to the following.

  First, the configuration of the imaging apparatus 100 according to the present embodiment will be described with reference to FIG. The imaging apparatus 100 includes an illumination device 110, a sample stage 131, an imaging optical system 140, a control unit 150, a monitor and input device 151, an imaging element 120, and an imaging stage 121.

  The illumination device 110 generates illumination light from the light source 111 and illuminates the sample 130 via a beam shaping system 112, a polarization state switching unit 113, and an illumination optical system (not shown). The polarization state switching unit 113 can switch the illumination light from the light source 111 to a desired polarization state. For example, the polarization state switching unit 113 includes a plurality of polarization elements and can select each of them. Here, it is assumed that the polarization state switching unit 113 is disposed at a position substantially conjugate with the pupil 141 of the imaging optical system 140. In FIG. 1, the illumination light is deflected by the mirror 115 to illuminate the sample 130, but the sample 130 may be directly illuminated without using the mirror 115.

  The sample stage 131 is connected to a moving mechanism (not shown) and is configured to support and drive the sample 130 to be imaged. The imaging element 120 is a photoelectric conversion element such as a CCD or CMOS sensor, and is supported by an imaging stage 121 that can move in the XY direction and the Z direction (optical axis direction). Note that the sample stage 131 and the imaging stage 121 are arranged so that the sample 130 and the imaging element 120 are optically conjugate.

  The imaging optical system 140 is an optical system that forms an image of the sample 130 illuminated by the illumination device 110. The imaging optical system 140 is an optical system that includes only a lens, an optical system that includes a concave mirror and a lens (catadioptric optical system), or a diffractive optical element. An optical system having the above can be used. When correction of chromatic aberration is necessary, a plurality of lenses made of glass materials having different dispersion values (Abbe values) can be used, or a diffractive optical element can be combined with a lens.

  The birefringent parallel plate 50 disposed on the optical path between the sample 130 and the image sensor 120 (on the optical axis of the imaging optical system 140) is made of a birefringent member. The birefringent parallel flat plate 50 may be disposed not only between the imaging optical system 140 and the imaging element 120 shown in FIG. 1 but also between the sample 130 and the imaging optical system 140, for example. Although the birefringent parallel plate 50 may be included in the imaging optical system 140, it is preferable that the imaging optical system 140 be detachable.

  The control unit 150 performs drive control of each unit, and controls the illumination device 110 based on the movement of the sample stage 131 and the imaging stage 121 and information input from the monitor and the input device 151. For example, the polarization state switching unit 113 is controlled in accordance with the input of the user of the imaging apparatus to switch the polarization state of the illumination light. Control information and other information by the control unit 150 are displayed on the monitor and input device 151.

  The sample stage 131, the imaging optical system 140, and the imaging stage 121 are provided, for example, on a stage barrel surface plate (not shown) supported by a base frame placed on a floor or the like via a damper or the like. It is done. Further, it is preferable that the imaging optical system 140 is designed based on one of the ordinary ray and the extraordinary ray on the birefringent parallel plate 50, and the imaging element 120 is disposed at the focal position of the ray as a reference.

  Next, an imaging method in the imaging apparatus 100 will be described in detail with reference to FIG. FIG. 2 is a schematic view of a main part showing the optical path in the imaging apparatus 100.

  First, the illumination light from the light source 111 is switched to a desired polarization state (details will be described later) by the polarization state switching unit 113 disposed in the vicinity of the pupil plane of the illumination optical system 114. Then, the sample 130 is illuminated via the illumination optical system 114, and the light beam from the sample 130 is divided into an ordinary ray and an extraordinary ray by the birefringent parallel plate 50 made of a birefringent member via the imaging optical system 140. The imaging optical system 140 forms an image with these light rays at two focal positions of positions 61 and 62 as shown in FIG. This is because the refractive index of the birefringent member takes different values for each of ordinary and extraordinary rays, and the value of each refractive index is determined by the type of birefringent member and the wavelength of the illumination light. As the thickness (length in the optical axis direction) of the birefringent parallel plate 50 increases, the distance (separation) between the two focal positions also increases. In this embodiment, considering the case where the refractive index for ordinary light is smaller than the refractive index for extraordinary light, ordinary light forms an image at position 61, and extraordinary light forms an image at position 62 behind it. Will form. Therefore, when an image sensor (not shown) is arranged at the position 61, the image is captured in a state where the image of the focused ordinary light beam and the image of the extraordinary light beam that is not focused are superimposed.

  In the conventional imaging method, the focus position is switched between the position 61 and the position 62 by controlling the polarization state of the illumination light, an image formed only with ordinary rays or abnormal rays is taken, and the obtained images are compared. The depth of focus was increased by selecting the one with less blur. However, for the thickness of the birefringent parallel plate 50 according to the present embodiment, the distance between the position 61 and the position 62 is sufficiently small, so that an image that can be sufficiently recognized even when the images at each position are superimposed is obtained. It will be possible. Therefore, when the polarization state switching unit 113 in the present embodiment wants to increase the depth of focus of the imaging optical system 140, the illumination light is made non-polarized (light that is not polarized), so that the ordinary ray and the extraordinary ray are made uniform. The images formed by each light beam are uniformly superimposed. As a result, an image with less blur can be acquired even if the images formed by the ordinary ray and the extraordinary ray are captured in a superimposed state. That is, the imaging apparatus 100 according to the present embodiment can increase the depth of focus without requiring complicated image processing. The same effect as that of non-polarized illumination can also be obtained by making the illumination light circularly polarized by the polarization state switching means 113.

Here, an appropriate thickness of the birefringent parallel plate 50 will be described. The separation of the focal position due to the ordinary ray and the extraordinary ray, as well as increase in the thickness d of the birefringent parallel plate 50, also by an increase in the difference Δn between the refractive index n _e for the refractive index n _o and an extraordinary ray with respect to the ordinary ray growing. Here, assuming that the wavelength of the illumination light is λ and the numerical aperture of the imaging optical system 140 is NA, if the separation of the focal position is about λ / NA ^{2 based} on subjective evaluation, the image at each focal position is superposed. It was confirmed that images with little blur could be acquired and the depth of focus could be expanded. Therefore, when a thickness d at which the focal position separation is λ / NA ² when a certain Δn is given is simulated, the following results are obtained. First, when the optical axis of the birefringent parallel plate 50 is parallel to the optical axis of the imaging optical system 140, the thickness d is 1.52 / Δn (λ / NA ² ) or less as shown in FIG. If so, the separation of the focal position becomes λ / NA ² or less. That is, it may be designed so as to satisfy the conditional expression of d ≦ 1.52 // n _o −n _e | (λ / NA ² ). When the optical axis of the birefringent parallel plate 50 is perpendicular to the optical axis of the imaging optical system 140, the thickness d is 1.27 / Δn (λ / NA ² ) or less as shown in FIG. If so, the separation of the focal position becomes λ / NA ² or less. That is, the conditional expression d ≦ 1.27 / | n _o −n _e | (λ / NA ² ) may be designed to be satisfied. Therefore, in the imaging apparatus 100 according to the present embodiment, it is desirable to design the thickness of the birefringent parallel plate 50 so as to be within the above range in consideration of the direction of the optical axis of the birefringent member (details will be described later). .

  As described above, when the image formed by the ordinary light beam at the position 61 and the image formed by the extraordinary light beam at the position 62 are overlapped and imaged, an image focused on one of the positions 61 or 62 is obtained. Compared with the case of imaging, the resolution of the acquired image is reduced. Therefore, when a high-resolution image of the sample 130 is to be acquired, it is necessary to set the polarization state so that the illumination light is not separated. Therefore, in the polarization state switching unit 113 according to the present embodiment, the illumination light is in the direction indicated by the arrow in FIG. 4A (radial direction) or the direction indicated by the arrow in FIG. 4B perpendicular to the radial direction. (Tangential direction) can be switched to one of the polarization states. These are polarization states called axially symmetric polarization in which the distribution of the polarization in the cross section of the light beam is linearly polarized light having a substantially uniform spatial distribution, and the distribution is radial or concentric. By using illumination light in such a polarization state, the occurrence of defocus aberration can be suppressed, and a high-resolution image can be acquired.

  As described above, according to the imaging apparatus 100 according to the present embodiment, the polarization state switching unit 113 switches the polarization state of the illumination light, thereby increasing the depth of focus image acquisition (focus depth expansion mode) or increasing the resolution. Image acquisition (high resolution mode) can be realized. For example, when it is desired to acquire an image in the depth of focus expansion mode, the imaging apparatus 100 sets the illumination light to non-polarized light or circularly polarized light when the user inputs the focus depth expansion mode selection using the input device 151, and the sample 130. Image. At this time, in the polarization state switching means 113, a depolarization plate, a fly-eye lens, a circular polarization filter, or the like can be selected. On the other hand, when the user wants to acquire an image in the high resolution mode, the user inputs the selection of the high resolution mode with the input device 151, so that the imaging device 100 transmits the illumination light in the radial direction shown in FIG. The sample 130 is imaged with polarization or tangential polarization shown in FIG. When the illumination light is visible light, a polarization state such as a liquid crystal element can be selected by the polarization state switching means 113 so that a polarization state in a radial direction or a tangential direction can be obtained.

  EXAMPLES Hereinafter, although the preferable Example of this invention is described, this invention is not limited to these Examples, A various deformation | transformation and change are possible within the range of the summary.

  In the present embodiment, a sample 130 is imaged with a light shielding member as shown in FIG. A coordinate system (x, y) in FIG. 5 represents the coordinates of each position of the sample 130 or the image of the sample 130. Here, in general, when observing a sample with an imaging device, the sample is covered with a cover glass (thickness of about 140 μm) to prepare a slide. In this embodiment, as shown in FIG. By using a birefringent member, this is regarded as a birefringent parallel plate 50. The light transmitted through the cover glass made of a birefringent member is separated into two light beams by birefringence, as in the case where the birefringent parallel plate 50 is disposed between the imaging optical system 140 and the image sensor 120 shown in FIG. Is done. That is, the image obtained by the image sensor 120 in FIG. 6 is obtained by integrating the images at the two focal positions. As described above, in the present embodiment, the configuration of the present invention can be easily realized without changing the design of the imaging optical system 140 by using the birefringent parallel plate 50 as the cover glass that covers the sample 130.

  In the illumination device 110, it is assumed that the light source 111 is a partially coherent light source, the illumination optical system 114 is a Kohler illumination system, and an intensity distribution as shown in FIG. 7 is formed on the pupil plane of the illumination optical system 114. The coordinate system (f, g) in FIG. 7 represents the coordinates of each position on the pupil plane of the illumination optical system 114. The pixel shown in white has an intensity of 1, and the pixel shown in black has an intensity of 0. is there. A circle drawn in gray represents the pupil of the illumination optical system 114. Each pixel shown in white represents a coherent point light source, and each coherent point light source is assumed to be incoherent with each other. That is, in this embodiment, the light source 111 (partial coherent light source) is regarded as a collection of a plurality of coherent point light sources that are incoherent with each other. In the illumination device 110 having such a light source 111 and illumination optical system 114, polarized illumination as shown in FIG. 4 can be realized by making each of the coherent point light sources be polarized in a desired direction.

Next, effects of the depth of focus expansion mode and the high resolution mode in the imaging apparatus 100 according to the present embodiment will be described. Here, a crystal having a thickness of 140 μm is used as the birefringent parallel plate (cover glass) 50, and the direction of the optical axis of the birefringent parallel plate 50 is set to be parallel to the optical axis of the imaging optical system 140. Think about the case. Note that the wavelength λ of the illumination light is 546.1 nm, and the NA of the imaging optical system 140 is 0.7. Under such conditions, the refractive index _{n o} for ordinary ray of the birefringence parallel plate 50 is 1.54617, the refractive index _{n e} for extraordinary ray is 1.55535, the separation of the focal position of each light beam each other, about 0.774 × (λ / NA ² )

First, in the case of the depth of focus expansion mode, the illumination light is made non-polarized or circularly polarized by the polarization state switching unit 113, so that two images having different focal positions by 0.774 × (λ / NA ² ) are superimposed. Get the image. In this state, as a result of measuring how the contrast of the image formed by the central opening of the sample 130 shown in FIG. 5 changes according to defocusing, a solid line shown in FIG. 8 was obtained. The contrast at the position of best focus (defocus is 0 nm) is about 82%, but the contrast decreases as the defocus increases, and the contrast is about 51% at the position of defocus 1125 nm.

  Here, in order to verify the effect of the present embodiment, in this imaging apparatus 100, a case where the cover glass is made of ordinary glass having no birefringence, that is, a case where the birefringent parallel plate 50 is not provided is considered. In this case, the same measurement as described above resulted in a dotted line shown in FIG. At the best focus position, the contrast is about 87%, and it can be seen that the resolution is higher than when the birefringent parallel plate 50 is provided. However, it can be seen that the rate of contrast reduction accompanying the increase in defocus is large, and the contrast is reduced to about 41% at a position where the defocus is 1125 nm.

  Therefore, when the depth-of-focus expansion mode is set in the imaging apparatus 100 according to the present embodiment, it is possible to suppress a decrease in contrast due to focus variation, and to increase the depth of focus of the imaging optical system 140. Strictly speaking, aberrations other than defocusing occur depending on the insertion position of the birefringent parallel flat plate 50, but the aberrations are as small as about 2 mλ and can be sufficiently ignored.

  On the other hand, when an image is acquired in the high resolution mode, the polarization state of the illumination light is changed to one of the polarization states shown in FIG. To form an image. For example, when the polarization state of the illumination light is set to the tangential polarization shown in FIG. 4B, the defocus dependence of the image contrast is as shown by the dotted line in FIG. 8, and the contrast at the best focus position is improved. doing. Therefore, when the high resolution mode is set in the imaging apparatus 100 according to the present embodiment, a high resolution image can be acquired.

  As described above, in the imaging apparatus 100 according to the present embodiment, the cover glass is made of the birefringent member (quartz), the direction of the optical axis thereof is set to be parallel to the optical axis of the imaging optical system 140, and the illumination light is supplied. The depth of focus can be expanded by using non-polarized light or circularly polarized light. Moreover, a high-resolution image can be acquired by making illumination light into radial polarization or tangential polarization.

In this embodiment, in the imaging apparatus 100 having the same configuration as that of the first embodiment, the direction of the optical axis of the birefringent parallel plate 50 (cover glass) is set to a direction perpendicular to the optical axis of the imaging optical system 140. Think. In the present embodiment, the same or equivalent components as those in the first embodiment are denoted by the same reference numerals, and the description thereof is simplified or omitted. In such a configuration, the separation of the focal position between the ordinary ray and the extraordinary ray is approximately 0.8967 × (λ / NA ² ). That is, compared with the case where the direction of the optical axis of the birefringent parallel plate 50 is parallel to the optical axis of the imaging optical system 140 in the first embodiment, the optical axis of the imaging optical system 140 is changed in this embodiment. It can be seen that the effect of birefringence appears more significantly when the direction is perpendicular to the direction.

  The result of performing the same measurement as in Example 1 as the depth of focus expansion mode is as shown by the solid line in FIG. 9, and the contrast at the best focus position is about 80%, but the defocus is at the position of 1125 nm. It can be seen that the contrast is 53%. On the other hand, the measurement result when the birefringent parallel plate 50 is not provided is as shown by a dotted line in FIG. 9, and the contrast is about 87% at the best focus position, and the contrast is about 41% at the defocus position of 1125 nm. To fall. Therefore, when the depth-of-focus expansion mode is set in the imaging apparatus 100 according to the present embodiment, it is possible to suppress a decrease in contrast due to focus variation, and to increase the depth of focus of the imaging optical system 140. Furthermore, by making the direction of the optical axis of the birefringent parallel plate 50 the direction perpendicular to the optical axis of the imaging optical system 140, the ratio of the contrast reduction accompanying the increase in defocusing can be made as compared with the first embodiment. Can be small.

  In addition, when the direction of the optical axis of the birefringent parallel plate 50 is set to a direction perpendicular to the optical axis of the imaging optical system 140, all the illumination lights can be switched to any polarization state shown in FIG. The ratio of ordinary and extraordinary rays to rays cannot be made equal. That is, since the imaging apparatus 100 according to the present embodiment does not enter the high resolution mode, when it is necessary to switch between the depth of focus expansion mode and the high resolution mode, the direction of the optical axis of the birefringent parallel plate 50 is set to the imaging optical system. The direction needs to be parallel to the 140 optical axes.

  As described above, in the imaging apparatus 100 according to the present embodiment, the cover glass is made of the birefringent member (quartz), the direction of the optical axis is set to the direction perpendicular to the optical axis of the imaging optical system 140, and the illumination light is emitted. The depth of focus can be expanded by using non-polarized light or circularly polarized light. Needless to say, the present invention is not limited to the above-described embodiments, and various modifications and changes can be made within the scope of the invention.

DESCRIPTION OF SYMBOLS 50 Birefringent parallel plate 100 Imaging device 110 Illumination optical system 111 Light source 130 Sample 113 Polarization state switching means 140 Imaging optical system 120 Imaging element

Claims (7)

An imaging apparatus comprising: an imaging optical system that images a sample illuminated by illumination light from a light source; and an imaging element that images the sample imaged by the imaging optical system,
A birefringent parallel plate disposed on an optical path between the sample and the imaging device and having birefringence with respect to the illumination light;
Polarization state switching means capable of switching the polarization state of the illumination light to non-polarized light or circularly polarized light, and radial polarized light or tangential polarized light with respect to the optical axis of the imaging optical system,
An imaging apparatus characterized in that the direction of the optical axis of the birefringent parallel plate is parallel to the optical axis direction of the imaging optical system. The thickness of the birefringent parallel plate in the direction of the optical axis is d, the wavelength of the illumination light is λ, the numerical aperture of the imaging optical system is NA, and the refractive indices of the birefringent parallel plate with respect to ordinary and extraordinary rays, respectively. a _n o, when the _{n e,}
d ≦ 1.52 // n _o −n _e | (λ / NA ² )
The imaging apparatus according to claim 1, wherein the following conditional expression is satisfied. The light source is a partially coherent light source composed of a plurality of coherent point light sources that are incoherent to each other,
The polarization state switching unit can switch the polarization state of the illumination light by switching the polarization state of a light beam from each of the plurality of coherent point light sources. Imaging device. An imaging apparatus comprising: an imaging optical system that images a sample illuminated by illumination light from a light source; and an imaging element that images the sample imaged by the imaging optical system,
A birefringent parallel plate disposed on an optical path between the sample and the imaging device and having birefringence with respect to the illumination light;
An imaging apparatus characterized in that the direction of the optical axis of the birefringent parallel plate is perpendicular to the optical axis direction of the imaging optical system. The thickness of the birefringent parallel plate in the direction of the optical axis is d, the wavelength of the illumination light is λ, the numerical aperture of the imaging optical system is NA, and the refractive indices of the birefringent parallel plate with respect to ordinary and extraordinary rays, respectively. a _n o, when the _{n e,}
d ≦ 1.27 / | n _o −n _e | (λ / NA ² )
The imaging apparatus according to claim 4, wherein the following conditional expression is satisfied.   The imaging apparatus according to claim 4, wherein the light source is a partially coherent light source including a plurality of coherent point light sources that are incoherent with each other.   The imaging device according to claim 1, wherein the birefringent parallel flat plate is a cover glass that covers the sample.

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