Patent Application
20220283058
Electron microscopy can be advantageously used to investigate the ultrastructure of biological samples such as cells and tissue, polymer resin samples, and crystalline samples such as inorganic substances. Two types of electron microscopes are known: scanning electron microscopes (hereinafter sometimes referred to as SEMs) and transmission electron microscopes (hereinafter sometimes referred to as TEMs).
In an electron microscope column, incident electrons are accelerated into, for example, epoxy resin-embedded samples (see
To advance different kinds of microscopy, researchers associated with this disclosure have developed a mechanically flexible and bendable conductive film that holds tissue, permits nanoscale cellular imaging, and eliminates charging artifacts resulting from the electron beam in scanning electron microscopes, transmission electron microscopes, etc. The conductive film can also be used for optical light microscopes to transmit light through the substrate for bright-field and fluorescence imaging.
In one embodiment of these films, graphene is uniformly coated on one side of a 0.5-mil Polyimide Kapton Film (No Additional Adhesive) 6.4 mm [¼ inch] wide×33 m [36 yd] long (PIT0.5N/6.4). Different electrical conductivity can be achieved by controlling the graphene coating's thickness, ranging from tens of nanometers to hundreds of nanometers. Typically, for about 5-10 nm thickness, a sheet resistance of less than 45 ohm/square is achieved.
The conductive fixation and graphene-based substrates make it possible to take advantage of inelastic scattering of electrons' capabilities to generate many signals (e.g., cathodoluminescence, Bremsstrahlung X-rays characteristic X-ray, secondary electrons, backscatter electrons, and Auger electrons) that scatter in an angular dependent manner. Additional research is exploiting their angle of deflection and their energy loss to gather chemical elemental information (e.g., C, N, Mg, P, Fe, Cu, and Zn) at the tissue, cellular, and subcellular levels. This enables mapping metal ions at electrical synapses on the nanometric-micrometric spatial scale, which is currently impossible to do.
Co-pending and commonly owned international PCT Application Serial No. PCT/US2019/013051, “Conductive Fixation of Organic Material,” filed Jan. 10, 2018 and U.S. Provisional Application Ser. No. 62/778,140 “Graphene Based Substrates for Imaging,” filed on Dec. 11, 2018, discuss the graphene substrates in more detail, and are incorporated herein as if set forth in their entireties.
To take advantage of high-resolution microscopies, the life sciences need better sample preparation workflows, reagents that will overcome charging and sample damage caused by electron beam-sample interactions in the electron microscope, and tools for accurate microscopic imaging in both two dimensional and three-dimensional views. These tools are set forth in detail herein.
Advancements in the kinds of slide mechanisms, or sample substrates, for sample collection have led to a need for more advanced sample collection on better slide surfaces than traditional slides that are known in the art. This disclosure provides a plurality of accessory tools for attachment to and use with an ultramicrotome, particularly for automatically collecting the sample sections onto a specialized sample surface, including but not limited to a graphene-based surface that is configured for accurate imaging under numerous kinds of microscopes and imagers. As an overview, this disclosure presents accessory tools for a microtome that aid in volume electron microscopy and specimen preparation. This disclosure provides supporting disclosure for at least four (4) different holders for four (4) different volume resin-based electron microcopy techniques (e.g., serial section scanning transmission electron microscopy (ssSTEM; see
In one embodiment, a system for sectioning resin-embedded cells and/or tissues with an ultramicrotome includes a modular reel-to-reel assembly comprising a reel-to-reel frame that connects to an upper region of a knife-stage used with the ultramicrotome, wherein the upper region of the knife-stage is adapted to connect the modular reel-to-reel assembly to the ultramicrotome. The modular reel-to-reel assembly further includes a feeder reel connected to a length of tape, wherein the feeder reel is rotatably coupled to the reel-to-reel frame; a take-up reel connected to the reel-to-reel frame and receiving the tape across the reel-to-reel frame; a feeder motor that drives rotation of the feeder reel; a take-up motor that drives rotation of the take-up reel; at least one cantilever arm position sensor connected to the modular reel-to-reel assembly and/or the ultramicrotome; and at least one electronic control unit controlling respective speeds of the feeder motor and the take-up motor, wherein the electronic control unit adjusts the respective speeds according to a cantilever arm position signals received from the at least one cantilever arm position sensor.
At least one embodiment further includes a knife stage adapter connecting the upper region of the knife stage to the reel-to-reel frame.
At least one embodiment further includes the feeder reel and the take-up reel made of aluminum wheels with respective hubs for wrapping the tape, the respective hubs having a calibrated circumference adapted to maintain a controllable speed and a settable tension of the tape extending between the feeder reel and the take-up reel.
At least one embodiment further includes the feeder reel and the take-up reel being made of aluminum and aluminum alloys having specified conductivity and specified magnetically shielding properties.
In other embodiments, the reel-to-reel frame is detachable from the upper region of the knife-stage without removing the feeder reel or the take-up reel or the tape.
In other embodiments, the feeder reel and the take-up reel are adapted to be re-attached to an in-situ scanning electron microscope reel-to-reel imaging system.
In other embodiments, the in-situ scanning electron microscope reel-to-reel imaging system is selected from secondary electron imaging systems, backscatter electron imaging systems, scanning transmission electron microscopy imaging systems.
In some non-limiting embodiments, an in-situ scanning electron microscope reel-to-reel imaging system is selected from quantitative measurement imaging systems comprising EDS and EELS systems.
In some embodiments, a specimen cantilever arm is connected to a specimen block chuck configured to hold a specimen block in engagement with an edge of a blade connected to a knife boat connected to the knife-stage connected to the ultramicrotome, wherein the at least one cantilever arm position sensor comprises at least a first cantilever arm position sensor attached to a base section of the ultramicrotome underneath a second cantilever arm position sensor attached to the specimen cantilever arm.
In non-limiting embodiments, the tape is a Kapton® polyimide tape positioned to receive thin, semi-thin, semi-thick, and thick resin sections from the edge of the blade connected to a knife boat, connected to a knife-stage, which is connected to the ultramicrotome.
In non-limiting embodiments, the tape may include a carbon coating.
In non-limiting embodiments, the carbon coating is a graphene coating.
In non-limiting embodiments, a graphical user interface is connected to the electronic control unit and configured to receive data entry for programming the electronic control unit.
The embodiments further include non-limiting concepts such as the data entry including speed selections for the feeder motor and the take-up motor and revolution selections for a hub of the feeder reel and the take up reel.
In one embodiment, at least one of the motors is an encoded stepper motor.
In at least one embodiment, a knife stage adapter includes a base portion adapted to bolt into an upper region of a ultramicrotome knife-stage; a platform region receiving at least one adjustment screw attaching the platform region to the base portion at a plurality of selectable heights; an adapter edge configured to receive a plurality of specimen substrate holders in a modular connection.
In at least one other embodiment, the knife stage adapter is configured to connect to a respective specimen substrate holder in the form of a reel to reel frame.
In at least one other embodiment, a respective specimen substrate holder is a coverslip vise, referred to also as a clip.
In at least one embodiment of a central specimen holder attachable to a knife stage adapter a respective specimen substrate holder is a triggered holding apparatus.
In at least one embodiment, a lever arm on the knife stage adapter is configured for arcuate movement relative to the base portion and configured to adjust a position of the specimen substrate holder along the Z-axis.
In at least one embodiment, a reel-to-reel frame for tape-based electron microscopy includes a feeder reel adapted to receive a length of tape, wherein the feeder reel is rotatably coupled to the reel-to-reel frame; a take-up reel adapted to receive the tape across the reel-to-reel frame; and wherein the reel-to-reel frame is configured to integrate with a knife-stage and connect to an ultramicrotome that transfers cut resin sections onto the tape.
In at least one embodiment, the reel to reel frame includes receptacles for bolting the reel-to-reel frame to a knife stage adapter, which connects to a knife stage on the ultramicrotome.
In at least one embodiment, the feeder reel and the take-up reel are exposed.
In at least one embodiment, the feeder reel and the take-up reel are modular and separable components that connect to the reel-to-reel frame.
In at least one embodiment, the reel-to-reel frame is configured to integrate with an ultramicrotome knife-stage.
In at least one embodiment, a system for receiving a cut sample from an ultramicrotome includes a knife stage adapter configured for removable attachment to a knife carrier on an ultramicrotome, wherein the knife stage adapter defines X, Y, and Z axes relative to the ultramicrotome; a vise attached to the knife stage adapter, wherein the vise is configured to hold a polymer coverslip in a position to receive the cut resin sections thereon; and a lever arm connected to the knife stage adapter and configured for arcuate movement about the knife stage adapter to position an end of the vise at selectable points along the Z axis and adjacent a specimen block on the ultramicrotome.
In one embodiment, the system places a cut resin section on the polymer coverslip.
In one embodiment, the cut resin section is one of a thin resin section, a semi-thin resin section, a semi-thick resin section, and a thick resin section.
In one embodiment, the vise is so dimensioned to hold a glass coverslip having a thickness in the range of 175 microns to 190 microns, the glass coverslip being compatible with optical microscopes.
In another embodiment, a scanning electron microscope stub holder is configured for use with an ultramicrotome to receive resin sections thereon and includes a substrate holder body defining a passageway with a first opening at one end of the holder body and a second opening extending from the first opening toward a second end of the holder body; a trigger that fits within the second opening, the trigger being actuated by a spring connected to the trigger and the holder body; a removable surface block configured to fit within the first opening of the holder body, the removable surface block held in place by the trigger biased by the spring.
In another embodiment, a fastening mechanism connects to a knife stage adapter that connects to a knife stage, wherein the fastening mechanism positions the removable surface block proximate the ultramicrotome.
In another embodiment, a substrate positioned across the surface block and held in place between sides of the surface block are connected to respective resin sections of the holder body and the trigger.
In another embodiment, a substrate positioned across the surface block includes a coated Kapton® polyimide layer or sheet.
In another embodiment, the substrate includes a carbon coated polyimide substrate.
In another embodiment, the stub holder further includes a graphene coated polyimide substrate.
In at least one other embodiment, an electronic controller allows for implementing electron microscopy on a sample received onto a coated polymer coverslip or onto a coated Kapton® polyimide tape, advanced from a feeder reel to a take-up reel connected to an ultramicrotome, and the controller includes a computer processor connected to computerized memory storing computer-implemented software implementing the steps. The steps include positioning the tape or the coverslip at an edge of a blade connected to a knife boat, which is connected to a knife-stage connected to the ultramicrotome, to collect thin, semi-thin, semi-thick, and/or thick resin sections after the sample is floated in water in the knife boat; receiving the sample onto the tape or the coverslip; and for the sample on the tape, advancing the tape to the take up reel.
In another embodiment, the electronic controller manages the feeder reel and the take up reel that are connected to an exposed reel-to-reel frame, and the computer implemented software further comprises computer implemented commands to position the exposed reel-to-reel frame in a Cartesian X, Y, Z coordinate system to receive cut resin sections on a coated Kapton® polyimide tape.
In another embodiment, the electronic controller is further configured for data communication with a control computer comprising a graphical user interface adapted to receive specifications for positioning the reel to reel frame.
In another embodiment, the electronic controller is connected to each of a feeder reel motor and a take up reel motor, wherein the computer implemented software comprises computer implemented commands to set at least one speed of the respective motors.
In another embodiment, the speed of the respective motors is coordinated by the electronic controller in conjunction with arcuate motion of a specimen block chuck holding a specimen block against a diamond knife-edge connected to the knife boat as controlled by the ultramicrotome, wherein the arcuate motion removes a resin section from the specimen block.
In another embodiment, the tracking software is stored in the memory, and the tracking software stores position data for the resin section placement on the tape.
In another embodiment, at least one data port receives a plurality of cantilever arm position signals received from a plurality of sample position sensors, and the cantilever arm position signals correspond to time and position data for each resin section placement on the tape.
In another embodiment, a position data registry stores the position data, time data, specimen dimensions, and tape dimensions in the memory to track placement of the resin section on the tape.
This disclosure further includes a method of transferring thin, semi-thin, semi-thick, and thick resin sections from the edge of a blade connected to a knife boat, connected to a knife-stage connected to an ultramicrotome, to a coated Kapton® polyimide tape, the method includes positioning a specimen block onto a nib of a specimen block chuck of the ultramicrotome; connecting a length of a coated Kapton® polyimide tape between a feeder reel and a take-up reel connected to a reel to reel frame; advancing the length of the coated Kapton® polyimide tape from the feeder reel to the take-up reel; positioning the coated Kapton® polyimide tape between the edge of a 35°-45° angle, 2-8 mm diamond blade and water in the knife boat; receiving cut resin sections from the water onto the coated Kapton® polyimide tape; and advancing the coated Kapton® polyimide tape.
The method embodiments further include embodiments that advance the coated Kapton® polyimide tape with respective stepper motors that drive rotation of the feeder reel and the take-up reel.
The method may further include embodiments that advance the coated Kapton® polyimide tape in iterative steps.
The method may further include embodiments that advance the coated Kapton® polyimide tape by continuously rotating the feeder reel and the take-up reel.
The method may further include programming an electronic control unit to advance the coated Kapton® polyimide tape.
The method may further include positioning the coated Kapton® polyimide tape at the edge of a 35°-45° angle, 6-8 mm diamond blade connected to a knife boat, connected to a knife-stage connected to the ultramicrotome, to collect thin, semi-thin, semi-thick, and thick resin sections after the respectively cut resin section is floated in the water in the knife boat.
The method may further include exposing the cut resin section to a spreading agent that unfolds the respectively cut resin section prior to collection of the section onto the coated Kapton® polyimide tape.
The method may further include exposing the respectively cut resin section to a chloroform vapor as the spreading agent.
The method may further include positioning an exhaust conduit proximate the ultramicrotome to control a concentration of chloroform vapor around the knife stage.
In yet another method of transferring samples from an ultramicrotome to a coated Kapton® polyimide tape and/or a coated polymer coverslip, the method steps may include positioning a cell and/or tissue resin embedded specimen block onto a nib of the ultramicrotome; connecting a coated Kapton® polyimide tape and/or a coated polymer coverslip to a knife-stage connected to the ultramicrotome; positioning a coated Kapton® polyimide tape and/or a coated polymer coverslip at the edge of a 35°-45° angle, 2-8 mm width diamond blade connected to a knife boat, connected to a knife-stage connected to the ultramicrotome, to collect thin, semi-thin, semi-thick, and thick resin sections after the cut resin section is floated in water in the boat; receiving the sample on a coated Kapton® polyimide tape and/or a coated polymer coverslip.
The method of transferring samples further includes bolting a knife stage adapter to the knife-stage; connecting the knife stage adapter to an electronic control unit configured to update respective adjustable positions of the knife stage adapter.
The method of transferring samples may further include embodiments, wherein the respective adjustable positions lie in a Cartesian X, Y, and Z plane defined by the knife stage adapter, and the electronic controller is configured to move the knife stage adapter in at least one direction in the Cartesian X, Y, Z plane.
The method of transferring samples may further include connecting a reel-to-reel frame with a feeder reel and a take up reel to the knife stage adapter.
The method of transferring samples may further include connecting a coverslip vise for array tomography to the knife stage adapter.
The method of transferring samples may include placing the electronic controller in data communication with a control computer having a graphical user interface and programming the electronic controller with the control computer.
The method of transferring a cut resin section of a specimen block attached to an ultramicrotome onto a coated Kapton® polyimide tape, the steps may include attaching a feeder reel wrapped with a tape to a reel-to-reel frame aligned with a specimen block on the ultramicrotome; placing at least part of the tape onto a tape guide across the reel-to-reel frame, attaching an end of the tape to a take-up reel; rotating the feeder reel and the take-up reel; advancing the tape from the feeder reel to the take-up reel along the tape guide and wrapping the tape around the take-up reel; and receiving the cut resin section onto the tape for storage on the take-up reel.
The method of transferring samples includes receiving a thin, semi-thin, semi-thick or thick resin sections onto a coated Kapton® polyimide tape.
The method of transferring samples includes using respective motors to rotate the feeder reel and the take-up reel.
The method of transferring samples include removing the reel-to-reel frame from the ultramicrotome without changing a position of the feeder reel, the take up reel, or the tape.
The method may further include storing the reel to reel frame on a stand.
These and other features, aspects, and advantages of the present invention will become apparent from the following description and the accompanying exemplary implementations shown in the drawings, which are briefly described below.
This disclosure describes an economical, compact device and system developed under the acronym “STAR”, which stands for “Scanning Transmission, Arraytome, Reel-to-Reel Microscopy.” Generally, this is a microscopy system that accommodates sample preparation with a commercial microtome and numerous specialized attachment accessories and tools that adapt the microtome for more efficient sample delivery. In one embodiment, STAR is a versatile, all-in-one ultramicrotome knife stage component accessory tool that is adaptable to any commercial ultramicrotome such as, e.g., but not limited to, Sorvall (MT 2; MT 2B; MT 5000; MT 6000); Leica (UC6; UC7); RMC Boeckeler (PowerTome) ultramicrotomes. The tools described herein accommodate the collection of hundreds to thousands of ultra-thin (thickness of 50 nm or less) and/or semi-thin (thickness above 50 nm), semi-thick (thickness between 51 nm and 100 nm) and thick (thickness between 101 nm and 200 nm) sample sections that can be automatically and continuously collected from a diamond knife edge onto a graphene-based, or other specialized material, surface. In some non-limiting embodiments, the sample collection surfaces may include coated 0.5-mil Polyimide [Kapton] Film No Adhesive 6.4 mm [¼″] wide×33 m [36 yd] long (PIT0.5N/6.4) tape for reel-to-reel microscopy, graphene coated coverslips, and graphene coated grids that collect samples cut from the microtome machinery. The references to graphene surfaces described herein are for example only and not limiting of the tools or uses of the tools described herein.
A plurality of accessory components provide sample sectioning and collection operations that accommodate reel to reel sectioning, serial sectioning, and array tomography sectioning. These components incorporate mechanical and electro-mechanical engineering to provide accessory tools for attachment to and use with an ultramicrotome 150, as shown in several of the figures of this disclosure.
By way of overview, this disclosure includes reference to one non-limiting commercial embodiment of an ultramicrotome illustrated in PRIOR ART
Options in the ultramicrotome, such as the example prior art of
Development of modern accessories to ultramicrotomes, as shown in one embodiment of this disclosure, includes a knife stage adapter that allows numerous accessories, systems and methods of specimen collection to be used with multiple versions of ultramicrotomes 150. The knife stage adapter 550, therefore, may be seen as one kind of adapter that connects specimen collection systems described herein to an ultramicrotome knife carrier 20 or general knife stage 158. A knife stage adapter 550 is shown in
In one non-limiting embodiment, the above described knife stage adapter 550 can hold (and release) a reel-to-reel frame 182 for specimen tape-based electron microscopy. The reel-to-reel frame is shown in detail in
In one non-limiting embodiment of the reel to reel frame 182 shown in
The feeder reel 175 and the take-up reel 177 may be, but are not limited to, machined aluminum wheels having respective hubs 173A, 173B for wrapping the specimen tape 190. The respective hubs 173A, 173B have a calibrated circumference, produced by precise machining often of aluminum, adapted to maintain a speed and a tension of the specimen tape 190 extending between the feeder reel 175 and the take-up reel 177. The aluminum material for the reels 175, 177, the hubs 173A, 173B, and the wheels on the hubs that secure the tape, is useful for its properties allowing precise control of conductivity and magnetic shielding.
The feeder reel 175 and the take-up reel 177 include aluminum compositions that are sufficient for withstanding temperatures inside of in-situ scanning electron microscopes that are equipped to use a reel-to-reel imaging system. Such temperatures in the environment of a scanning electron microscope may rise to the range of 1400 degrees Celsius to 1500 degrees Celsius. As noted above and illustrated in
This disclosure presents several different kinds of central specimen holders 182, 300, 435 that can be positioned relative to an ultramicrotome knife stage 158 and specimen cantilever arm 152 to receive sliced specimens on a number of different slides, or substrates. The modular, separable components of the knife stage adapter 550 and the numerous kinds of central specimen holders 182, 300, 435 allow for complete versatility in specimen collection and specimen analysis with other kinds of equipment.
The versatility of these operations is shown in a system for preparing cut samples with an ultramicrotome 150. In
As shown in
The system of
As noted above in regard to
This disclosure accounts for multiple, non-limiting versions of the firmware for the control environment, illustrated in
One non-limiting version of the firmware is considered for the knife stage 550 with an additional slicer arm monitor module. This module monitors the periodical movement of a slicer arm with the help of a sensor. The information of the slicer arm speed controls the tape speed, which is important to maintain a constant distance between the sliced samples.
The second version of the firmware may be used for the tape coating hardware of companion disclosures. Besides the tape control module, another module for controlling a third step motor, is included. This motor module handles the coating of the tape by turning at a constant speed. The tape heater module is measuring the temperature of a heater block by an RDT sensor. An implemented PID control maintains a set temperature of the heating block, whose output controls an electric heating element attached to the heating block.
Using various embodiments of this disclosure to operate a scanning electron microscopy procedure is the third version of the firmware. In addition, for the tape control module, an analog joystick is attached to the controller. The joystick can control the tape speed, tape direction as well as sample position during manual mode. This is important to center the sample for the microscope.
In non-limiting example embodiments of a controller 700, there are three operations. All three operations have the tape control in common, which can have two different modes. If the source spool motor has no encoder attached, the user has to set the initial revolutions of the sink-spool and source-spool manually on the interface. With an encoder, only the revolution of the sink-spool has to be set. The controller will calculate the revolution of the source-spool automatically. For the knife stage operations, the knife-stage operation can be run with two different modes, depending if the slicer monitor is activated. If the knife-stage runs without the slicer monitor, the user can set the tape speed and the tape direction. With the activated slicer monitor, instead of tape speed and direction the user can set sample distance. In this mode, the tape direction always goes from source-spool to sink-spool. The tape speed is calculated by the sample distance and the monitored speed of the slicer arm.
When the tape is played back for use with scanning electron microscopy (SEM) operation, the user first has to center the first sample manually with the joystick. On the interface then the user has to set sample distance. This information has to be taken from the interface of the knife-stage controller. Another important setting is the scan time of the sample, which pauses the tape during sample scan.
In a coating operation of companion embodiments to this disclosure, there are three settings. The first setting is the speed of the coating motor. This determine in combination with the tape speed, how much material will be coated on the tape. The other two settings are heating temperature and heating exposure time. The heating exposure time will determine the speed of the tape. The tape direction is fixed in this mode and can only go from source-spool to sink-spool.
The figures, such as but not limited to
As noted above, the knife stage adapter 550 is also accessible for attaching other central specimen holders to the ultramicrotome 150 for sample collection. In this embodiment, a sample substrate holder for receiving a cut sample from an ultramicrotome 150 includes a clip type holder, also called a vise 435, for specimen collection in preparation for array tomography. In one non-limiting embodiment, a knife stage adapter 550 is configured for removable attachment to a knife stage 158 on an ultramicrotome 150, wherein the knife stage adapter 550 defines X, Y, and Z axes relative to the ultramicrotome. As shown in
A sample substrate holder may also encompass a trigger holder as illustrated in
Any one of the above noted central sample substrate holders, including the reel-to-reel embodiments, the clip embodiments, or the trigger embodiments can be used to perform methods of sample collection. Along these lines, a method of transferring samples from an ultramicrotome to a specimen tape includes positioning a sample block onto a nib 814 (
The system and apparatuses of this disclosure are configured to provide a maximum flexibility for a method of transferring samples from an ultramicrotome to a specimen substrate. As noted above, the ultramicrotome may be used with a number of different kinds of central specimen holders, including but not limited to a reel-to-reel system, a trigger holder system, and a clip mechanism. Each of these different embodiments enable a method of positioning a sample block onto a nib 814 of the ultramicrotome 150; connecting a specimen substrate to a central specimen holder 185, 300, 435; positioning the specimen substrate between the sample block and a knife stage adapter 550 connected to the ultramicrotome 150, wherein the knife stage adapter 158 receives a portion of the sample block against a knife edge 439 to slice at least one sample from the sample block; and receiving the sample on the sample substrate.
As noted above, the ultramicrotome 150 has a knife stage 158 that may operate with a knife stage adapter 550 to receive the central specimen holder in adjustable positions and connecting the central specimen holder to an electronic control unit configured to update respective adjustable positions of the central specimen holder. The respective adjustable positions lie in a Cartesian X, Y, and Z plane defined by the knife stage adapter 550, and the electronic controller 700 is configured to move the central specimen holder in at least one direction in the Cartesian X, Y, Z plane. The method can continue by (i) connecting a reel to reel frame 182 with a feeder reel 175 and a take up reel 177 as the central specimen holder, (ii) connecting a clipping apparatus 435 for array tomography as the central specimen holder, or (iii) connecting a triggered holder apparatus 300 for array tomography as the central specimen holder. By placing the electronic controller 700 in data communication with a control computer having a graphical user interface 154 and programming the electronic controller 700 with the control computer, the methods herein may be automated.
Automated methods of this disclosure include a method of transferring a cut sample section of a specimen block attached to an ultramicrotome 150 onto an electron microscopy specimen tape 190 by attaching a feeder reel 175 wrapped with the specimen tape 190 to a reel-to-reel frame 182 aligned with the specimen block on the ultramicrotome 150; placing at least part of the specimen tape 190 on a tape guide 180 across the reel-to-reel frame 182, attaching an end of the specimen support tape 190 to a take-up reel 177; rotating the feeder reel 175 and the take-up reel 177; advancing the specimen tape 190 from the feeder reel 175 to the take-up reel 177 along the tape guide 180 and wrapping the specimen tape 190 around the take-up reel 177; and receiving the cut sample section onto the substrate strip for storage on the take-up reel 177. The automated methods enable receiving a semi thick or thin ultramicrotome cut sample onto the sample tape 190 while simultaneously using respective motors 1410, 1420 to rotate the feeder reel 175 and the take-up reel 177 in a controlled way of advancing the specimen tape 190. Upon completion, the method as disclosed herein optionally includes removing the reel-to-reel frame 182 from the ultramicrotome 150 without disrupting the feeder reel 175, the take up reel 177, or the specimen tape 190.
The automated methods described above are enabled in part by utilizing an electronic controller 700 for implementing tape-based electron microscopy on a sample received onto a sample support tape 190. The electronic controller 700 may move the specimen tape 190 that is advanced from a feeder reel 175 to a take-up reel 177 connected to an ultramicrotome 150. The electronic controller typically includes a computer processor 200 connected to computerized memory storing computer-implemented software implementing programmable, computerized steps of a method. The method includes positioning the specimen support tape 190 between a sample block and a knife stage connected to the ultramicrotome, wherein the knife stage receives a portion of the sample block against a knife edge to slice at least one sample of the sample block; receiving the sample on the specimen support tape 190; and advancing the specimen support tape.
As shown in the figures, the feeder reel 175 and the take up reel 177 are connected to an exposed reel to reel frame 182, and the computer implemented software further comprises computer implemented commands to position the exposed reel to reel frame in a Cartesian X, Y, Z coordinate system to receive the sample on the specimen support tape. The computer implemented software comprises computer implemented commands to set at least one speed of the respective motors. The speed of the respective motors is coordinated by the electronic controller 700 in conjunction with arcuate motion 21 of the sample block against the knife edge 439 as controlled by the ultramicrotome 150.
The terminology used herein is for the purpose of describing particular implementations only and is not intended to be limiting of the invention. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “comprises” and/or “comprising,” when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.
The corresponding structures, materials, acts, and equivalents of all means or step plus function elements in the claims below are intended to include any structure, material, or act for performing the function in combination with other claimed elements as specifically claimed. The description of the present invention has been presented for purposes of illustration and description, but is not intended to be exhaustive or limited to the invention in the form disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the invention. The implementation was chosen and described in order to best explain the principles of the invention and the practical application, and to enable others of ordinary skill in the art to understand the invention for various implementations with various modifications as are suited to the particular use contemplated.
The figures utilize an exemplary computing environment in which example embodiments and aspects may be implemented. The computing device environment of
Numerous other general purpose or special purpose computing devices environments or configurations may be used. Examples of well-known computing devices, environments, and/or configurations that may be suitable for use include, but are not limited to, personal computers, server computers, handheld or laptop devices, multiprocessor systems, microprocessor-based systems, network personal computers (PCs), minicomputers, mainframe computers, embedded systems, distributed computing environments that include any of the above systems or devices, and the like.
Computer-executable instructions, such as program modules, being executed by a computer may be used. Generally, program modules include routines, programs, objects, components, data structures, etc. that perform particular tasks or implement particular abstract data types. Distributed computing environments may be used where tasks are performed by remote processing devices that are linked through a communications network or other data transmission medium. In a distributed computing environment, program modules and other data may be located in both local and remote computer storage media including memory storage devices.
In its most basic configuration, a computing device typically includes at least one processing unit and memory. Depending on the exact configuration and type of computing device, memory may be volatile (such as random-access memory (RAM)), non-volatile (such as read-only memory (ROM), flash memory, etc.), or some combination of the two.
Computing devices may have additional features/functionality. For example, computing device may include additional storage (removable and/or non-removable) including, but not limited to, magnetic or optical disks or tape. Such additional storage is illustrated in
Computing device typically includes a variety of computer readable media. Computer readable media can be any available media that can be accessed by the device and includes both volatile and non-volatile media, removable and non-removable media.
Computer storage media include volatile and non-volatile, and removable and non-removable media implemented in any method or technology for storage of information such as computer readable instructions, data structures, program modules or other data. Memory, removable storage, and non-removable storage are all examples of computer storage media. Computer storage media include, but are not limited to, RAM, ROM, electrically erasable program read-only memory (EEPROM), flash memory or other memory technology, CD-ROM, digital versatile disks (DVD) or other optical storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices, or any other medium which can be used to store the desired information and which can be accessed by computing device. Any such computer storage media may be part of computing device.
Computing device 200 may contain communication connection(s) that allow the device to communicate with other devices. Computing device may also have input device(s) such as a keyboard, mouse, pen, voice input device, touch input device, etc. Output device(s) such as a display, speakers, printer, etc. may also be included. All these devices are well known in the art and need not be discussed at length here.
It should be understood that the various techniques described herein may be implemented in connection with hardware components or software components or, where appropriate, with a combination of both. Illustrative types of hardware components that can be used include Field-programmable Gate Arrays (FPGAs), Application-specific Integrated Circuits (ASICs), Application-specific Standard Products (ASSPs), System-on-a-chip systems (SOCs), Complex Programmable Logic Devices (CPLDs), etc. The methods and apparatus of the presently disclosed subject matter, or certain aspects or portions thereof, may take the form of program code (i.e., instructions) embodied in tangible media, such as CD-ROMs, hard drives, or any other machine-readable storage medium where, when the program code is loaded into and executed by a machine, such as a computer, the machine becomes an apparatus for practicing the presently disclosed subject matter.
This application claims priority to and incorporates by reference four United States Provisional patent applications including U.S. Application Ser. No. 62/883,918, U.S. Application Ser. No. 62/883,496, U.S. Application Ser. No. 62/891,067, and U.S. Application Ser. No. 62/893,534.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US20/45233 | 8/6/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62883496 | Aug 2019 | US | |
| 62883918 | Aug 2019 | US | |
| 62891067 | Aug 2019 | US | |
| 62893534 | Aug 2019 | US |
