microscope

Abstract

A microscope includes: a sample stage for mounting a sample; a light source for illuminating the sample when the sample is mounted on the sample stage; a detector; a first objective lens disposed on one side of the sample stage; a second objective lens disposed on an opposite side of the sample stage; a first set of optical elements defining a first light path from the first objective lens to the detector; and a second set of optical elements defining a second light path from the second objective lens to the detector. The first objective lens and the second objective lens have a common optical axis and are configured to image the sample mounted on the sample stage in a common focal plane. In addition, the first objective lens is a high-power objective lens and the second objective lens is a low-power objective lens. Providing such a microscope configuration enables simultaneous viewing of the sample at high and low magnifications, and / or allows rapid switching between high-power images and low-power images, for example, to provide nearly simultaneous viewing at two magnifications.

Description

Technical Field

[0001] The present invention relates to optical microscopy. In particular, but not exclusively, the present invention relates to confocal optical microscopy. Background Art

[0002] Many conventional optical microscopes include multiple objectives with different magnifications. These objectives are mounted in a turret, which allows them to be alternately positioned in the microscope's optical path, allowing the sample to be viewed at different magnifications. This allows the user to identify an area of ​​interest at a relatively low magnification and then view that area at a higher magnification.

[0003] However, conventional optical microscopes of the type described above cannot view a sample at two magnifications simultaneously because only one objective can be placed in the optical path at a time. They also do not allow for rapid switching between a high-power (magnification) image and a low-power image, for example, to provide near-simultaneous viewing at two magnifications. These represent technical problems with known optical microscopes.

[0004] A similar problem arises with confocal microscopy, in which an image is generated by scanning a sample using a high-magnification objective, but a low-magnification objective can also be used to orient and locate regions of interest in the sample. Again, both objectives cannot be used simultaneously, and therefore, high-magnification and low-magnification images cannot be captured simultaneously or nearly simultaneously. This also represents a technical problem with known confocal microscopy.

[0005] In some confocal microscopes, light from a light source is focused onto a single spot on the sample. Light emitted from this spot is then collected by an objective lens (e.g., by reflection or fluorescence) and focused onto a detector. By scanning the sample and detecting changes in the intensity of the light coming from the sample, an image of the sample can be constructed. However, because the sample needs to be scanned, real-time imaging is not possible.

[0006] In an alternative confocal system (e.g., as described in US Pat. No. 6,144,489), a mask is provided for encoding light incident on a sample and decoding light emitted from multiple regions of the sample. This simultaneously generates a confocal image and a non-confocal image of the sample. By removing the non-confocal image from the confocal image, out-of-focus blur can be removed, thereby providing an improved confocal image. However, this confocal system again suffers from the above-mentioned technical problems, specifically, because it cannot capture both high-magnification and low-magnification images simultaneously.

[0007] A similar confocal microscope system is described in US6687052. Summary of the Invention

[0008] It is an object of the present invention to provide a microscope which overcomes or alleviates one or more of the above problems, or provides a useful alternative thereto.

[0009] According to the present invention, there is provided a microscope comprising:

[0010] a sample stage for mounting samples;

[0011] a light source for illuminating the sample when the sample is mounted on the sample stage;

[0012] detector;

[0013] a first objective lens disposed on one side of the sample stage;

[0014] a second objective lens disposed on an opposite side of the sample stage;

[0015] a first set of optical elements defining a first optical path from the first objective lens to the detector;

[0016] a second set of optical elements defining a second optical path from the second objective lens to the detector;

[0017] wherein the first objective lens and the second objective lens have a common optical axis and are configured to image a sample mounted on a sample stage in a common focal plane,

[0018] The first objective lens is a high-power objective lens, and the second objective lens is a low-power objective lens (ie, the first objective lens has a higher magnification than the second objective lens).

[0019] Providing such a microscope configuration enables simultaneous viewing of a sample at high and low magnifications, and / or allows rapid switching between high and low magnification images, for example to provide near-simultaneous viewing at two magnifications.

[0020] The microscope may further include a beam splitter installed in the first optical path and the second optical path. The beam splitter may include a semi-transparent mirror or include a spatial light modulator including a plurality of transmissive portions and a plurality of non-transmissive portions.

[0021] For example, the beam splitter can be in the form of a rotatable mask comprising a plurality of transmissive portions and a plurality of non-transmissive portions. Such a beam splitter enables the first and second light paths to be split into several portions as they pass through the detector. This is useful in many applications where a portion of the light beam needs to be selected and processed to generate a desired image.

[0022] For example, the non-transmissive portion of the spatial light modulator can be reflective, and the spatial light modulator can be configured to split a first light path into a first transmitted light path and a first reflected light path, and to split a second light path into a second transmitted light path and a second reflected light path, wherein the first transmitted light path coincides with the second reflected light path, and the first reflected light path coincides with the second transmitted light path. The transmitted light path and the reflected light path can then be used to construct an image.

[0023] For example, the detector may include a first detector portion and a second detector portion, wherein the first detector portion is configured to receive transmitted light and generate transmitted image data, and the second detector portion is configured to receive reflected light and generate reflected image data, or the second detector portion is configured to receive transmitted light and generate transmitted image data, and the first detector portion is configured to receive reflected light and generate reflected image data.

[0024] In one configuration, the first detector portion is configured to receive light from the first transmitted light path or a coincident second reflected light path, and the second detector portion is configured to receive light from the second transmitted light path or a coincident first reflected light path. The microscope may further include an image processor connected to receive image data from the detectors and configured to subtract reflected image data from the transmitted image data to generate confocal image data for one or both of the first objective and the second objective. This configuration can enable laser-free confocal microscopy that includes simultaneous or near-simultaneous high-magnification and low-magnification imaging.

[0025] In such an arrangement, the light source may comprise, for example, an LED lamp.

[0026] Furthermore, the light source may be configured to direct light toward the sample stage through the beam splitter for illuminating a sample mounted on the sample stage through the first objective lens and / or the second objective lens. This arrangement may provide a confocal system having similar functionality to that described in US6144489.

[0027] The beam splitter can be used as a mask to encode the light incident on the sample and decode the light emitted from multiple regions of the sample. This will simultaneously generate a confocal image and a non-confocal image of the sample. By subtracting the non-confocal image from the confocal image, out-of-focus blur can be removed, thereby providing an improved confocal image. However, in contrast to US6144489, the confocal system described herein can capture high-magnification images and low-magnification images simultaneously and / or nearly simultaneously.

[0028] The microscope may also include a shutter mechanism for switching between the first objective lens and the second objective lens. Thus, the user can view either a low-magnification image or a high-magnification image without making any other changes to the microscope's optical configuration. This makes it very simple for the user to switch between a low-magnification image for orientation and a high-magnification image of a specific area of ​​interest within the specimen. Furthermore, by rapidly switching the shutter mechanism, a pair of high-magnification and low-magnification images can be captured substantially simultaneously.

[0029] The first objective lens can be moved relative to the sample stage along an optical z-axis, and the microscope can further include a z-stack controller configured to drive the first objective lens along the optical z-axis and capture a series of images while the first objective lens is driven along the optical z-axis. Such z-stack controllers are known. The difference here is that the z-stack controller is configured to capture a series of images while the first objective lens is moving, without requiring the first objective lens to be stopped when capturing images. This can significantly shorten the time period used to capture the z-stack of images. For example, in one configuration, components of the sample stage itself move only in the x-y plane relative to the optical z-axis to cause the sample to move in the x-y plane. The camera is driven along the z-axis, thereby acquiring the z-stack while the camera is continuously driven in the z direction.

[0030] The sample stage itself can include a transparent base on which the sample can be mounted and a slide mechanism for sliding the sample around on the top surface of the transparent base. This configuration enables the desired x–y motion of the sample while maintaining the desired z positioning of the sample. It also allows the sample to be viewed through two objective lenses on either side of the sample stage.

[0031] The microscope may also include an integrated data storage unit for storing image data. As described herein, large amounts of image data can be generated very quickly by the microscope. Thus, it has been found advantageous to provide an integrated data storage unit for storing image data. This data can then be processed within the microscope and / or transmitted to an external device (e.g., a computing device such as a laptop, desktop computer, tablet computer, or smartphone) for image processing and / or viewing. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Embodiments of the present invention will now be described by way of example with reference to the accompanying drawings, in which:

[0033] Figure 1 is a schematic diagram of a prior art confocal microscope apparatus, and

[0034] Figure 2 is a schematic diagram of a first embodiment of a confocal microscope apparatus according to the present invention. DETAILED DESCRIPTION

[0035] Figure 1 Schematic diagram of a prior art confocal microscope apparatus as described in US Pat. No. 6,687,052. The apparatus comprises a light source 1 with an associated collimating lens 2, which directs light toward a beam splitter 3, such as a half-silver mirror, a polarizing beam splitter combined with a quarter-wave plate, or a dichroic beam splitter. From the beam splitter 3, the light is reflected through a mask 6 and a microscope objective 4 onto a sample O mounted on a sample stage 5.

[0036] The mask 6 comprises a spatial modulator that modulates the light from the light source 1 to the sample O. Typically, the mask modulates the intensity of the light passing through it, however, alternatively it may modulate the phase or polarisation of the light.

[0037] In one embodiment, the mask 6 includes a pattern of transmissive and opaque or reflective areas that modulate the intensity of light passing through the mask. The mask is configured to allow the transmissive and reflective areas to be moved around to adjust the illumination pattern falling on the sample O. This can be achieved, for example, by arranging a pattern of transmissive and reflective areas on a rotating disk. Alternatively, a spatial light modulator comprising an adjustable micromirror array can be used.

[0038] Light emitted from the sample O (e.g., by reflection or fluorescence) is captured by the objective lens 4 and focused back onto the mask 6. The light that passes through the transmissive portion of the mask 6 then passes through the beam splitter 3 and is focused by the lens 8 onto a first detector 7 (e.g., a CCD detector). The first detector 7 captures a first image of the sample O.

[0039] The mask 6 is arranged at a small angle (e.g. a few degrees) to the optical axis X of the objective lens 4. As a result, light reflected from the reflective part of the mask is focused by the lens 9 onto a second detector 11 which captures a second image of the sample.

[0040] The images captured by the first detector 7 and the second detector 11 are sent to an image processing device 10 , which is configured to generate a composite image based on the images captured by the first detector 7 and the second detector 11 .

[0041] In use, light emitted from the sample O is focused by the microscope objective 4 back onto the mask 6, which transmits only the light falling on the transmission areas of the mask. A transmission image is captured by the first detector 7. The image captured by the first detector 7 (referred to herein as the positive image) comprises a combination of a conventional image Iconv superimposed with a confocal image Iconf, where the confocal image Iconf is generated by light emitted from the focal plane of the microscope objective 4.

[0042] At the same time, light emitted from other parts of the sample O (i.e., from other planes of the sample) is reflected by the reflective portions of the spatial pattern on the rear surface of the mask 6 and is focused onto the second detector 11. Thus, the second detector 11 captures a second image (referred to herein as a negative image) comprising the conventional image Iconv from which the confocal image Iconf is subtracted.

[0043] The positive image captured by the first detector 7 and the negative image captured by the second detector 11 are transmitted to the image analyzer 10 which subtracts the negative image from the positive image, i.e.:

[0044] (Iconv+Iconf)-(Iconv-Iconf).

[0045] The resulting image is a confocal image as described in US6687052 from which the out-of-focus conventional image has been removed.

[0046] Figure 2 A confocal microscope according to an embodiment of the present invention is shown. As will be described in more detail below, the optical arrangement is generally similar to Figure 1 The optical arrangement of a prior art confocal microscope is shown in . However, in the present invention, two microscope objectives 20, 22 are provided, which are mounted on opposite sides of a sample holder 24, thereby allowing two images of the sample to be captured simultaneously (or nearly simultaneously), provided that the sample is at least partially transparent.

[0047] In one embodiment, the microscope objective lenses 20 and 22 have different magnifications, with the first microscope objective lens 20 comprising a high-power objective lens and the second microscope objective lens 22 comprising a low-power objective lens providing a wide field of view.

[0048] exist Figure 2 In the illustrated embodiment, a high-power optical path 25a from high-power objective 22 to detector 26 is defined by a set of optical components, which in this embodiment include a shutter 28a, a mirror 30, a lens 32, and a beam splitter 33 including a mask 34. In this embodiment, mask 34 comprises a rotating disk having a pattern of transmissive and reflective regions. However, other types of masks, such as a spatial light modulator, may also be used.

[0049] Likewise, the low-magnification optical path 25 b from the low-magnification objective lens 22 to the detector 26 is defined by a set of optical elements, which in this embodiment include a shutter 28 b , a mirror 30 , a lens 32 , and a mask 34 .

[0050] It should be noted that both the high-magnification optical path 25 a and the low-magnification optical path 26 b pass through the mask 34 between the corresponding objective lenses 20 , 22 and the detector 26 .

[0051] It should also be noted that Figure 2 The arrangement of the mirror 30 and lens 32 shown in FIG is purely illustrative and is not intended to be limiting. In practice, the layout of these components may be varied significantly without changing the functional operation of the microscope.

[0052] The confocal microscope also includes a light source 36 that introduces a light beam along the high-magnification optical path 25a or the low-magnification optical path 25b, for example, via a beam splitter 38. In this embodiment, light from the light source 36 is introduced along the high-magnification optical path at a location between the detector 26 and the mask 34. The light source 26 can be, for example, a low-coherence light source (e.g., a light emitting diode (LED), an incandescent lamp, or an arc lamp), or a coherent light source (e.g., a laser).

[0053] In use, light from the light source 36 passes through the mask 34 and is focused by the high power objective lens 22 onto a focal plane within the sample O. As previously described with respect to Figure 1 As described in the prior art microscope shown in , light is encoded by a mask 34 to produce a spatial pattern that is imaged onto a sample in a sample holder 24 .

[0054] The coded light emitted back from the sample O (e.g., by reflection or fluorescence) is focused by the high-power objective 20 onto a mask 34, which transmits only the light falling on the transmission areas of the mask. A transmission image is captured on a first portion 26 a of the detector 26. This is a positive image that includes a conventional image Iconv superimposed with a confocal image Iconf generated by light emitted from the focal plane of the high-power objective 20.

[0055] At the same time, light emanating from other parts of the sample O (i.e., not in the focal plane of the microscope objective 20) is reflected by the reflective portion of the spatial pattern of the mask 34 and is focused onto the second portion 26b of the detector 26. The detector 26 thus captures a negative image comprising the conventional image Iconv from which the confocal image Iconf has been subtracted.

[0056] The positive and negative images captured by detector 26 are transmitted to image analyzer 40 which subtracts the negative image from the positive image, thereby producing a high-power confocal image from which the out-of-focus conventional image has been removed.

[0057] Alternatively, a low-magnification confocal image can be captured by detecting light passing through the low-magnification objective lens 22 and along the low-magnification optical path 25b. To capture a low-magnification image, the first shutter 28a in the high-magnification optical path 25a is closed and the second shutter 28b in the low-magnification optical path 25b is opened.

[0058] The sample O is illuminated by light reflected from the second side 34b of the mask 34. As previously described, the spatial pattern carried by the mask 34 includes a plurality of transmissive portions and a plurality of reflective portions. Thus, the light reflected from the second side 34b is encoded by the reflective portions in a manner similar to the way in which the light transmitted through the mask 34 is encoded by the transmissive portions. The coded light reflected from the mask is focused by the low-power objective lens 22 onto the sample O, thereby forming an image having the spatial pattern in a focal plane within the sample O. Preferably, the focal plane of the low-power objective lens 22 is coplanar with the focal plane of the high-power objective lens 20, so that the two objective lenses 20, 22 image the same plane within the sample O.

[0059] Light emitted from the sample is focused by low-power objective lens 22 onto mask 34, which transmits only the light that falls on the transmissive areas of the mask. Light that falls on the reflective areas of the mask is decoded by the spatial pattern of the mask. This light forms a low-power positive image on first portion 26a of detector 26.

[0060] Light transmitted through the transmissive portion of the mask 34 is focused on the second portion 26b of the detector, thereby forming a negative low-magnification image. The positive low-magnification image and the negative low-magnification image are transmitted to the image analyzer 40, which subtracts the negative image from the positive image to form a low-magnification confocal image from which the out-of-focus conventional image has been removed.

[0061] As will be apparent, by sequentially opening the first shutter 28a and the second shutter 28b, the user can view either a low-magnification confocal image or a high-magnification confocal image without making any other changes to the optical configuration of the microscope. This makes it very simple for the user to switch between a low-magnification confocal image for orientation and a high-magnification confocal image of a specific area of ​​interest within the sample. Furthermore, by rapidly and alternately switching the first shutter 28a, 28b between the open and closed configurations, a pair of high-magnification and low-magnification images can be captured substantially simultaneously.

[0062] Although the above embodiments relate to confocal microscopy, the present invention is also applicable to non-confocal microscopy, where it allows two different images of a sample, such as a high-magnification image and a low-magnification image, to be captured simultaneously or nearly simultaneously. This can be achieved by using a beam splitter (e.g., a half-silvered mirror) instead of a spatial modulator forming the mask 34.

[0063] In this arrangement, light from a light source 36 falls on a beam splitter including a mask 34. A portion of this light will be transmitted through the mask 34, and a portion will be reflected. The transmitted light will follow a high-power optical path to the high-power objective lens 20, which will focus the light onto the sample O. The light emitted from the sample O will then be captured by the high-power objective lens 20 and will pass through the mask 34 to be focused onto the first portion 26 a of the detector 26, thereby forming a high-power image of the sample O. Note that any light reflected from the mask 34 will not be captured, and the first image is a conventional image rather than a confocal image.

[0064] Similarly, a low-magnification image can be captured using the low-magnification objective lens 22. To capture the low-magnification image, the first shutter 28a is closed to block light traveling along the high-magnification optical path 25a, and the second shutter 28b is opened to allow light to travel along the low-magnification optical path 25b. Light reflected from the mask 34 will reach the low-magnification objective lens 22 along the low-magnification optical path 25b, which will focus the light onto the sample O. The light emitted from the sample O will then be captured by the low-magnification objective lens 22 and will pass through the mask 34 to be focused onto the second portion 26b of the detector 26, thereby forming a low-magnification image of the sample O. Again, this image is a conventional image rather than a confocal image.

[0065] The examples described herein are to be understood as illustrative examples of embodiments of the present invention. Other embodiments and examples are contemplated. Any feature described with respect to any example or embodiment may be used alone or in combination with other features. Furthermore, any feature described with respect to any example or embodiment may also be used in combination with one or more features of any other example or embodiment, or any combination of any other examples and embodiments. Furthermore, equivalents and modifications not described herein may also be employed within the scope of the present invention as defined in the claims.

Claims

1. A microscope comprising: A sample stage, used for mounting a sample; a light source for illuminating the sample when the sample is mounted on the sample stage; detector; a first objective lens disposed on one side of the sample stage; a second objective lens disposed on an opposite side of the sample stage; a first set of optical elements defining a first optical path from the first objective lens to the detector; and a second set of optical elements defining a second optical path from the second objective lens to the detector; wherein the first objective lens and the second objective lens have a common optical axis and are configured to image a sample mounted on the sample stage in a common focal plane, wherein the first objective lens is a high-power objective lens, and the second objective lens is a low-power objective lens, The microscope further comprises a beam splitter installed in the first optical path and the second optical path, the beam splitter comprises a spatial light modulator, and the spatial light modulator comprises a plurality of transmission parts and a plurality of non-transmission parts. wherein the non-transmissive portion of the spatial light modulator is reflective, wherein the spatial light modulator is configured to split the first light path into a first transmission light path and a first reflection light path, and split the second light path into a second transmission light path and a second reflection light path, and The first transmission light path is consistent with the second reflection light path, and the first reflection light path is consistent with the second transmission light path.

2. The microscope according to claim 1, in, The spatial light modulator includes a rotatable mask including a plurality of transmissive portions and a plurality of non-transmissive portions.

3. The microscope according to claim 1, in, The detector includes a first detector portion and a second detector portion, wherein the first detector portion is configured to receive transmitted light and generate transmitted image data, and the second detector portion is configured to receive reflected light and generate reflected image data, or the second detector portion is configured to receive transmitted light and generate transmitted image data, and the first detector portion is configured to receive reflected light and generate reflected image data.

4. The microscope according to claim 3, in, The first detector portion is configured to receive light from the first transmitted light path or the identical second reflected light path, and the second detector portion is configured to receive light from the second transmitted light path or the identical first reflected light path.

5. The microscope according to claim 3 or 4, in, The microscope also includes an image processor connected to receive image data from the detector and configured to subtract the reflected image data from the transmitted image data to produce confocal image data for one or both of the first objective and the second objective.

6. The microscope according to any one of claims 1 to 4, in, The microscope further includes a shutter mechanism for switching between the first objective lens and the second objective lens.

7. The microscope according to any one of claims 1 to 4, in, The light source is configured to direct light toward the sample stage through the beam splitter for illuminating a sample mounted in the sample stage through the first objective lens and / or the second objective lens.

8. The microscope according to claim 7, in, In use, light from the light source passing through the beam splitter is directed to the sample along the first optical path via the first objective lens, and / or light from the light source reflected by the beam splitter is directed to the sample along the second optical path via the second objective lens.

9. The microscope according to any one of claims 1 to 4, in, The first objective lens is capable of moving along an optical z-axis relative to the sample stage, and the microscope includes a z-stack controller, which is configured to drive the first objective lens along the optical z-axis and capture a series of images when the first objective lens is driven along the optical z-axis, and the z-stack controller is configured to capture the series of images while the first objective lens is moving without stopping the first objective lens when capturing the images.

10. The microscope according to any one of claims 1 to 4, in, The microscope further comprises an integrated data storage unit for storing image data.

11. The microscope according to any one of claims 1 to 4, in, The sample stage includes a transparent base and a sliding mechanism. The sample can be mounted on the transparent base, and the sliding mechanism is used to make the sample slide around on the top surface of the transparent base.

Images