WO2018089578A1

WO2018089578A1 - Gridtape imaging stage

Info

Publication number: WO2018089578A1
Application number: PCT/US2017/060770
Authority: WO
Inventors: Brett Jesse GRAHAM; Wei-Chung Allen LEE; David Grant Colburn HILDEBRAND
Original assignee: President And Fellows Of Harvard College
Priority date: 2016-11-10
Filing date: 2017-11-09
Publication date: 2018-05-17

Abstract

An imaging system is directed to electron microscopy of collected samples and includes a feed reel configured to initially store a tape with the collected samples. The imaging system further includes a pick-up reel configured to receive the tape from the feed reel, and an imaging-path element extending between the feed reel and the pick-up reel. The imaging-path element has a first end near the feed reel and a second end near the pick-up reel, the first end being connected to the second end via an intermediate section of the imaging-path element along which the tape translates linearly from the feed reel to the pick-up reel.

Description

GRIDTAPE IMAGING STAGE

CROSS-REFERENCE TO RELATED APPLICATION(S)

[0001] This application claims priority and the benefit of U.S. Provisional Patent Application No. 62/420,550, filed November 10, 2016, which is hereby incorporated by reference herein in its entirety.

GOVERNMENT SUPPORT

[0002] This invention was made with government support under grant no. R21NS085320 and grant no. T32HL007901, each grant awarded by the National Institutes of Health. The government has certain rights in the invention.

FIELD OF THE INVENTION

[0003] The present invention relates generally to high, nanometer-resolution electron microscopy ("EM") and, more particularly, to a stage for imaging samples on a transmission EM-compatible tape substrate.

BACKGROUND OF THE INVENTION

[0004] The ability to collect and image ever increasing numbers of serial tissue sections has proved invaluable in neuroscience, biomedical, and life sciences fields. Similar collection and imaging processes of samples are useful for applications in materials science, nanotechnology, semiconductor, and other fields. Emerging tape-based section collection and processing techniques have the benefit of maintaining section order and are amenable to automated processing that reduces errors and speeds up dataset generation. By way of example, transmission electron microscopy ("TEM") offers high resolution, high signal-to- noise, and fast acquisition rates, but is typically hampered by the laborious requirements of manually collecting specimens onto separate sample holding substrates and subsequently manually exchange samples in the EM for image acquisition. This is primarily due to the lack of previous technologies to automatically collect EM specimens onto a sample substrate compatible with transmitted electron detection.

[0005] One alternative approach to such laborious manual techniques is covering a tape- based substrate to produce a tape sandwich, such as disclosed in U.S. Patent No. 8,366,857 ("the '857 Patent"), titled "Methods, Apparatus And Systems For Production, Collection, Handling, And Imaging Of Tissue Sections." However, this approach has not become a useful solution as it is inefficient and overly complex, with an original reliance on an overly complex automatic taping lathe-microtome cutting and deficient imaging system. By way of example, problems associated with the system of the '857 Patent include a lack of compatibility with a tape-based substrate that allows imaging of every sample region (without having to skip every other sample region), a lack of compatibility with a perforated tape that operates without a cover tape, and a lack of compatibility for allowing a flexible density of sections per length of tape. Another problem of the imaging system of the '857 Patent is that it is overly complex in design and operation.

[0006] The present disclosure is directed at providing a microscope stage for handling samples collected onto a tape substrate that solves the above and other needs.

SUMMARY OF THE INVENTION

[0007] According to one aspect of the present invention, an imaging system is directed to EM of collected samples, such as tissue samples, and includes a feed reel configured to initially store a tape with the collected samples. The imaging system further includes a pickup reel configured to receive the tape from the feed reel, and an imaging-path element extending between the feed reel and the pick-up reel. The imaging-path element has a first end near the feed reel and a second end near the pick-up reel, the first end being connected to the second end via an intermediate section of the imaging-path element along which the tape translates from the feed reel to the pick-up reel.

[0008] According to another aspect of the present invention, a method is directed to imaging collected samples in an EM system. The method includes storing a tape with the collected samples on a feed reel, and translating the tape linearly from the feed reel to a pickup reel along an imaging-path element. The imaging-path has a first end near the feed reel and a second end near the pick-up reel. The method further includes feeding the tape through an entry port of an electron microscope located along an intermediate section of the imaging- path element between the first end and the second end. The tape exits through an exit port of the electron microscope that is located, for example, at 180 degrees relative to the entry port.

[0009] According to yet another aspect of the present invention, an imaging stage is directed to EM of collected samples and includes a feed reel containing a tape with the collected samples. The imaging stage further includes a pick-up reel configured to subsequently receive the tape from the feed reel, the pick-up reel being spaced on an opposite side of an imaging electron beam from the feed reel. The imaging stage further includes an imaging-path channel extending between the feed reel and the pick-up reel, the imaging-path channel forming a path along which the tape is transferred from the feed reel to the pick-up reel. The imaging-path channel includes an intermediate section configured to be received through a pair of microscope ports oriented at various angles (e.g., 180 degrees) relative to each other. The imaging stage also includes a pair of pinch drives positioned between the feed reel and the pick-up reel that are configured to control tape tension and move the tape as it is transferred along the imaging-path channel.

[0010] Additional aspects of the invention will be apparent to those of ordinary skill in the art in view of the detailed description of various embodiments, which is made with reference to the drawings, a brief description of which is provided below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIG. 1 is a perspective view of an imaging system for EM.

[0012] FIG. 2A is a top view of the imaging system of FIG. 1.

[0013] FIG. 2B is a cross-sectional front view along lines "2B-2B" of FIG. 2A.

[0014] FIG. 2C is a side view of the imaging system of FIG. 1.

[0015] FIG. 3 shows the imaging system of FIG. 2B with exemplary component identifiers.

[0016] FIG. 4A is a front view of the imaging system of FIG. 1.

[0017] FIG. 4B is a cross-sectional top view along lines "4B-4B" of FIG. 4 A.

[0018] FIG. 5 is an enlarged view of a piezoelectric stack and a tension sensor of the imaging system of FIG. 1.

[0019] FIG. 6 is an enlarged view of a pickup rest of the imaging system of FIG. 1.

[0020] FIG. 7 is perspective view of the imaging system of FIG. 1 with an illustrated exemplary embodiment of a microscope column at a sample level.

[0021] While the invention is susceptible to various modifications and alternative forms, specific embodiments have been shown by way of example in the drawings and will be described in detail herein. It should be understood, however, that the invention is not intended to be limited to the particular forms disclosed. Rather, the invention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the appended claims.

DETAILED DESCRIPTION

[0022] While this invention is susceptible of embodiment in many different forms, there is shown in the drawings and will herein be described in detail preferred embodiments of the invention with the understanding that the present disclosure is to be considered as an exemplification of the principles of the invention and is not intended to limit the broad aspect of the invention to the embodiments illustrated. For purposes of the present detailed description, the singular includes the plural and vice versa (unless specifically disclaimed); the words "and" and "or" shall be both conjunctive and disjunctive; the word "all" means "any and all"; the word "any" means "any and all"; and the word "including" means "including without limitation." Where a range of values is disclosed, the respective embodiments include each value between the upper and lower limits of the range.

[0023] Referring to FIG. 1, an imaging system 100 for EM of collected samples includes a number of components that allow the safe unspooling, nanometer-scale movement, and re- spooling of tape-based substrates within an electron microscope 200 (illustrated in FIG. 9). The imaging system 100, according to the illustrated embodiment, is in the form of an imaging stage that includes two reel drives 102a, 102b connected via torque-limiting slip clutches to two respective reels 104a, 104b that reside within a vacuum environment. The reel drives 102a, 102b contain reels 104a, 104b, and reel drive motors 106a, 106b. The reel drive motors 106a, 106b maintain a constant torque and keep a tape 105 (illustrated in FIG. 2B), with collected samples, tensioned on the reels 104a, 104b. According to one example, the collected samples are tissue samples.

[0024] The reel drives 102a, 102b include a feed reel drive 102a and a pick-up reel drive 102b. The feed reel drive 102a includes a feed reel drive motor 106a and a feed reel 104a, which is configured to initially store the tape 105 with the collected samples. The pick-up reel drive 102b includes a pick-up reel drive motor 106b and a pick-up reel 104b, which is configured to receive the tape 105 from the feed reel 104a. The imaging system 100 further includes a feed pinch drive 108a with a pinch drive motor 110a and a pick-up pinch drive 108b with a pinch drive motor 110b for dispensing or collecting the tape 105 during movements, such as section-to-section movements

[0025] An imaging-path element 112 extends between the feed reel 104a and the pick-up reel 104b, having a first end 112a near the feed reel 104a and a second end adjacent to a pickup rest 103 and near the pick-up reel 104b. The first end 112a of the imaging-path element 112 is connected to the second end 112b via an intermediate section 112c along which the tape 105 translates from the feed reel 104a to the pick-up reel 104b.

[0026] The imaging-path element 112 further has a sample imaging slot 112d positioned in the intermediate section 112c. The sample imaging slot 112d is dimensioned and sized to allow pass-through of an electron beam when using the microscope 200 to image the collected samples. According to one example, as referred to below, the imaging-path element 112 is a tape clamp in the form of a C-shaped cross-section slot channel that rests on a ledge on the feed side of the imaging system 100, near the feed reel 104a.

[0027] Referring to FIGs. 2A-2C, the feed pinch drive 108a further includes a passive feed idle roller 114a positioned near a controllable feed drive roller 116a, and the pick-up pinch drive 108b further includes a passive pick-up idle roller 114b positioned near a controllable pick-up drive roller 116b. The pinch drives 108a, 108b dispense or collect the tape 105 by trapping the tape 105, between the controllable drive rollers 116a, 116b and the respective passive idle rollers 114a, 114b.

[0028] The imaging system 100 further includes a tension sensor 118 located between the pinch drives 108a, 108b. The tension sensor 118 ensures that the tape is not over-tensioned during movement and imaging of the tape 105.

[0029] The imaging system 100 also includes a stack of positioning stages 120 that is also located between the pinch drives 108a, 108b. The positioning stages 120 allow for nanometer-scale movements needed for imaging of the samples collected on tape 105. The tape 105 is coupled to the positioning stages 120 with the tape clamp 112.

[0030] Referring to FIG. 3, the imaging system 100 translates the tape 105 from the feed reel 104a, through the tape clamp 112, to the pick-up reel 104b, and uses the coordinated action of the pinch drives 108a, 108b at entry port 202a and exit port 202b of a microscope column 201. The accuracy and precision of large tape translations require the tape 105 to be placed under a consistent, empirically-derived tension that is measured by the tension sensor 118, which according to one example is an on-stage load cell. The tape 105 is further moderated by the relative motion of the pinch drives 108a, 108b.

[0031] The imaging system 100 is further configured to increase movement precision for micro-positioning of the tape 105 during high-magnification montage imaging. By way of example, to meet the accuracy and precision required for effective montage imaging, the positioning stages 120 includes at least one 2-axis piezoelectric stage that translates the tape clamp 112.

[0032] The pinch drives 108a, 108b sufficiently de-tension the tape 105 to allow freedom of movement by the piezoelectric stage 120 to provide accurate and precise tape micro- positioning. Corresponding de-tensioning of the tape 105 enable both (a) large magnitude translations of the tape 105 (e.g., >1 millimeter) and (b) piezo-mediated micro-positioning that facilitates high-magnification montage imaging. As such, the tape 105 is adjustable by both (1) large-scale bi-directional translation of the tape 105, and (2) multi-directional micro- positioning.

[0033] Referring to FIGs. 4A and 4B, the tape 105 is adjustable in in at least two directions, direction "X" and direction "Y", which are, respectively, along an X-axis and along a Y-axis. The tape 105 is optionally further adjustable in a direction Z," along a Z-axis (as illustrated in FIG. 2B). According to some examples, the accuracy and precision of large magnitude translations of the tape 105 via the pinch drives 108a, 108b is in the range of about +/- 1 micrometer under normal tensioned conditions, and in the range of about +/- 3 micrometers when de-tensioning or re-tensioning occurs. Furthermore, the accuracy and precision is sufficient for advancing through successive sample regions on the tape 105 when the tape 105 is held at a recommended tension. By way of further example, the translation speed of the tape 105 is at a minimum speed of about 4.8 millimeters/second.

[0034] Referring to FIG. 5, the stack of positioning stages 120 include, according to one example, an X-stage 120a and an Y-stage 120b that are located near the tension sensor 118. The stack of positioning stages 120 are optionally communicatively coupled to a controller 130 in which one or more searching algorithms are stored. According to an example, an increase in accuracy is achieved through coordinate stage movement and the one or more searching algorithms. By way of example, under the control of a piezo-mediated micro- positioning system, accuracy and precision of movement is increased by using a single X translation back and forth at a new Y position to relieve any binding mechanical forces that may generate Y movement variances.

[0035] Referring to FIG. 6, the pick-up rest 103 functions as a vibration-damping element that helps reduce or eliminate vibrations in a sample. The vibrations are generally caused by environmental or stage movements before images are acquired. The reduction or elimination of vibrations, in turn, helps achieve the high-resolution and signal quality generally provided in a TEM. For example, following movements of the tape 105, image acquisition is delayed by a set "settling time" that, when high, significantly limits the overall acquisition rate. To reduce the "settling time," the imaging system 100 moves only the tape clamp 112 and a small portion of the tape 105 that contains the sample being imaged.

[0036] Additionally, the tape clamp 112 extends through the column 200 (as illustrated in FIG. 3) and rests on a vibration-damping surface 103a of the pick-up rest 103. By supporting the tape clamp 112 at both ends, the potential cantilever configuration of the tape clamp 112 is avoided, thereby reducing or eliminating undesired vibrations typically associated with cantilever configurations. [0037] Referring to FIG. 7, the imaging system 100 is located in a vacuum environment in an example context containing the electron microscope 200, which is illustrated at a sample level. The electron microscope 200 includes the microscope column 201 through which the tape 105 enters (via the entry port 202a) and exits (via the exit port 202b). According to this example, the entry and exit ports 202a, 202b are located at 180 degrees relative to each other. However, in other examples, the entry and exit ports 202a, 202b are located at other angles relative to each other. Thus, the imaging system 100 enables movement of the tape 105 from the feed reel drive 102a to the pick-up reel drive 102b through the microscope column 201 located in the imaging path between the two reel drives 102a, 102b. An electron beam 204 of the microscope 200 represents the imaging location of each imaged sample region on the tape 105.

[0038] Some exemplary benefits of the imaging system 100 is that it is compatible with tape-based substrates and that it can be retrofitted onto many existing TEMs (which are already operating in laboratories and imaging facilities). Another exemplary benefit of the imaging system 100 is that it stands apart from other proposed tape-based substrate imaging stages by allowing imaging of every sample region on the tape. In contrast, current tape- based imaging stages require the imaging to skip every other sample region (such as the system of the '857 Patent).

[0039] The imaging system 100 is further beneficial because it allows the operation of the imaging stage without a cover tape or any other tape sandwich. As such, these benefits of the imaging system 100 allow for a higher density of sections per length of the tape 105, and result in a greatly simplified design and operation.

[0040] In reference to imaging of large datasets or sample libraries, yet another benefit of the imaging system 100 is that it reduces or eliminates prior limitations in the ability to quickly and reliably move samples between imaging locations of a sample. Thus, such limitations associated with stock TEM sample positioners, which are not optimized for fast movement and settling, are overcome by the imaging system 100 by using fast nanometer- positioners (e.g., the positioning stages 120) that are capable of speeds of several millimeters per second. Thus, the imaging system 100 greatly improves movements speed, reducing it from seconds to tens of milliseconds.

[0041] Each of these embodiments and obvious variations thereof is contemplated as falling within the spirit and scope of the claimed invention, which is set forth in the following claims. Moreover, the present concepts expressly include any and all combinations and subcombinations of the preceding elements and aspects.

Claims

CLAIMS What is claimed is:

1. An imaging system for electron microscopy (EM) of collected samples, the imaging system comprising:

a feed reel configured to initially store a tape with the collected samples;

a pick-up reel configured to receive the tape from the feed reel; and

an imaging-path element extending between the feed reel and the pick-up reel, the imaging-path element having a first end near the feed reel and a second end near the pick-up reel, the first end being connected to the second end via an intermediate section of the imaging-path element along which the tape translates from the feed reel to the pick-up reel.

2. The imaging system of claim 1, wherein the imaging-path element is a tape translation device in the form of a C-shaped slot channel that includes a tape clamp for stabilizing the tape during image data acquisition.

3. The imaging system of claim 1, further comprising a vibration-damping element with a vibration-damping surface, at least a portion of the imaging-path element being positioned on top of and in contact with the vibration-damping surface.

4. The imaging system of claim 3, wherein the first end of the imaging-path element is in direct contact with the vibration-damping surface.

5. The imaging system of claim 3, wherein the second end of the imaging-path element is in contact with the vibration-damping surface.

6. The imaging system of claim 3, wherein the second end of the imaging-path element is supported on the vibration-damping element.

7. The imaging system of claim 1, further comprising at least one positioning stage connected to the imaging-path element such that movement of the positioning stage causes adjustment of the imaging-path element.

8. The imaging system of claim 7, wherein the positioning stage includes translation motion in at least two directions, along an X-axis and along a Y-axis.

9. The imaging system of claim 7, wherein the positioning stage is moved by a piezoelectric device.

10. The imaging system of claim 1, further comprising an electron microscope positioned between the feed reel and the pick-up reel along the intermediate section of the imaging-path element, the electron microscope having two ports including an entry port and an exit port, the entry port being located on the opposite side relative to the exit port, the intermediate section of the imaging-path element being inserted through the two ports such that the tape is fed through the entry port and exits through the exit port.

11. The imaging system of claim 1, further comprising a feed pinch-drive and a pick-up pinch-drive, the feed pinch-drive being located near the feed reel and the pick-up pinch-drive being located near the pick-up reel, the feed pinch-drive and the pick-up pinch-drive controlling tension of the tape as it translates along the imaging-path element.

12. The imaging system of claim 1, further comprising respective controllable drive rollers and passive idle rollers coupled to each of the feed reel and the pick-up reel, the controllable drive rollers and the passive idle rollers controlling respective motion of the feed reel and the pick-up reel.

13. The imaging system of claim 1, further comprising a tension sensor configured to detect tension of the tape between the first end and the second end of the imaging-path element.

14. A method for imaging collected samples in an electron microscope, the method comprising:

storing a tape with the collected samples on a feed reel;

translating the tape linearly from the feed reel to a pick-up reel along an imaging-path element, the imaging-path having a first end near the feed reel and a second end near the pick-up reel; and

feeding the tape to an electron microscope located along an intermediate section of the imaging-path element between the first end and the second end.

15. The method of claim 14, further comprising obtaining images of the collected samples via the electron microscope.

16. The method of claim 14, further comprising reducing vibrations of the tape by placing the imaging-path element in contact with a vibration-damping surface.

17. The method of claim 14, further comprising adjusting the tape in a plurality of directions independent of the electron microscope.

18. An imaging stage for electron microscopy (EM) of collected samples, the imaging stage comprising:

a feed reel containing a tape with the collected samples;

a pick-up reel configured to subsequently receive the tape from the feed reel, the pickup reel being spaced from the feed reel;

an imaging-path channel extending between the feed reel and the pick-up reel, the imaging-path channel forming a path along which the tape is transferred from the feed reel to the pick-up reel, the imaging-path channel including an intermediate section configured to be received through a pair of microscope ports located opposite to each other; and

a pair of pinch drives positioned between the feed reel and the pick-up reel and configured to control tape tension and move the tape as the tape is transferred along the imaging-path channel.

19. The imaging stage of claim 18, further comprising a first vibration-damping element and a second vibration-damping element, the first vibration-damping element having a first vibration-damping surface on which a first end of the imaging-path channel is supported near the feed reel, the second vibration-damping element having a second vibration-damping surface on which a second end of the imaging-path channel is supported near the pick-up reel.

20. The imaging stage of claim 18, further comprising one or more positioning stages connected to the imaging-path channel, at least one of the one or more positioning stages being movable in a plurality of directions, movement of the one or more positioning stages causing adjustment of the imaging-path channel.