U.S. patent application number 17/633421 was filed with the patent office on 2022-09-08 for apparatus and methods for ultramicrotome specimen preparation.
The applicant listed for this patent is THE UNIVERSITY OF KANSAS. Invention is credited to Allen HASE, Christian HORNHUBER, Eduardo ROSA-MOLINAR, Robert YOUNG.
Application Number | 20220283058 17/633421 |
Document ID | / |
Family ID | 1000006418386 |
Filed Date | 2022-09-08 |
United States Patent
Application |
20220283058 |
Kind Code |
A1 |
ROSA-MOLINAR; Eduardo ; et
al. |
September 8, 2022 |
APPARATUS AND METHODS FOR ULTRAMICROTOME SPECIMEN PREPARATION
Abstract
A plurality of accessory components for use in respective reel
to reel sectioning, serial sectioning, and array tomography
sectioning described herein incorporate mechanical and
electro-mechanical engineering to provide accessory tools for
attachment to and use with a microtome, as shown in several of the
figures of this disclosure.
Inventors: |
ROSA-MOLINAR; Eduardo;
(Lawrence, KS) ; HASE; Allen; (Lawrence, KS)
; YOUNG; Robert; (Lawrence, KS) ; HORNHUBER;
Christian; (Lawrence, KS) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE UNIVERSITY OF KANSAS |
Lawrence |
KS |
US |
|
|
Family ID: |
1000006418386 |
Appl. No.: |
17/633421 |
Filed: |
August 6, 2020 |
PCT Filed: |
August 6, 2020 |
PCT NO: |
PCT/US20/45233 |
371 Date: |
February 7, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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62883496 |
Aug 6, 2019 |
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62883918 |
Aug 7, 2019 |
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62891067 |
Aug 23, 2019 |
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62893534 |
Aug 29, 2019 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 2001/364 20130101;
G01N 1/06 20130101; H01J 37/20 20130101; G01N 1/2806 20130101 |
International
Class: |
G01N 1/06 20060101
G01N001/06; G01N 1/28 20060101 G01N001/28; H01J 37/20 20060101
H01J037/20 |
Claims
1. A system for sectioning resin-embedded cells and/or tissues with
an ultramicrotome, the system comprising: 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, and wherein the
modular reel-to-reel assembly further comprises: 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.
2. The system of claim 1, further comprising a knife stage adapter
connecting the upper region of the knife stage to the reel-to-reel
frame.
3. The system of claim 1, wherein the feeder reel and the take-up
reel comprise aluminum wheels comprising respective hubs for
wrapping the tape, the respective hubs having a calibrated
circumference adapted to maintain a speed and a tension of the tape
extending between the feeder reel and the take-up reel.
4. The system of claim 3, wherein the feeder reel and the take-up
reel are made of aluminum and aluminum alloys comprising specified
conductivity and specified magnetically shielding properties.
5. The system of claim 1, wherein 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.
6. The system of claim 5, wherein 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.
7. The system of claim 6, wherein 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.
8. The system of claim 6, wherein the in-situ scanning electron
microscope reel-to-reel imaging system is selected from
quantitative measurement imaging systems comprising EDS and EELS
systems.
9. The system of claim 1, further comprising a specimen cantilever
arm 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.
10. The system of claim 9 wherein the tape is a Kapton.RTM.
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.
11. The system of claim 10, wherein the tape further comprises a
carbon coating.
12. The system of claim 11, wherein the carbon coating is a
graphene coating.
13. The system of claim 1, further comprising a graphical user
interface connected to the electronic control unit and configured
to receive data entry for programming the electronic control
unit.
14. The system of claim 13, wherein the data entry comprises speed
selections for the feeder motor and the take-up motor.
15. The system of claim 14, wherein at least one of the motors is
an encoded stepper motor.
16.-58. (canceled)
59. A method of transferring a cut resin section of a specimen
block attached to an ultramicrotome onto a coated Kapton.RTM.
polyimide tape, the method comprising: 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.
60. The method of claim 59, further comprising receiving a thin,
semi-thin, semi-thick or thick resin sections onto a coated
Kapton.RTM. polyimide tape.
61. The method of claim 59, further comprising using respective
motors to rotate the feeder reel and the take-up reel.
62. The method of claim 61, further comprising removing the
reel-to-reel frame from the ultramicrotome without changing a
position of the feeder reel, the take up reel, or the tape.
63. The method of claim 62, further comprising storing the reel to
reel frame on a stand.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] 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.
BACKGROUND
[0002] 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).
[0003] In an electron microscope column, incident electrons are
accelerated into, for example, epoxy resin-embedded samples (see
FIG. 1). A number of interactions between the accelerated electrons
and atoms contained within the resin-embedded sample result in
elastic and inelastic scattering of electrons (known as the
electron interaction volume; see FIG. 1). A number of signals
generated (i.e., secondary electrons, backscattered electrons,
cathodoluminescence, auger electrons, characteristic X-rays, and
Bremsstrahlung X-rays) can be used for high-resolution electron
microscopic imaging of ultrastructural features of cell and tissue
organelles.
[0004] 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.
[0005] 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 [1/4 inch] wide.times.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.
[0006] 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.
[0007] 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.
[0008] 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.
BRIEF SUMMARY OF THE DISCLOSURE
[0009] 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 FIGS. 27A,
27B); array tomography (Arraytome [AT]; see FIG. 31] and
reel-to-reel microscopy [rrM; see FIG. 8]). In one non-limiting
embodiment, each of these configurations (ssSTEM, AT, rrM) are
disclosed herein with the appropriate accessory tools that
accommodate the microtome depositing ultra-thin (50 nm or less)
and/or semi-thin (above 50 nm) sections on the Kapton graphene
coated tape, coverslips, stubs, and grids. These sample collection
surfaces can be post-stained or immuno-stained using fluorescence
and/or chromogens of immunogold and/or immunogold derivative
labels.
[0010] 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.
[0011] At least one embodiment further includes a knife stage
adapter connecting the upper region of the knife stage to the
reel-to-reel frame.
[0012] 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.
[0013] 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.
[0014] 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.
[0015] 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.
[0016] 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.
[0017] 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.
[0018] 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.
[0019] In non-limiting embodiments, the tape is a Kapton.RTM.
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.
[0020] In non-limiting embodiments, the tape may include a carbon
coating.
[0021] In non-limiting embodiments, the carbon coating is a
graphene coating.
[0022] 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.
[0023] 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.
[0024] In one embodiment, at least one of the motors is an encoded
stepper motor.
[0025] 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.
[0026] 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.
[0027] In at least one other embodiment, a respective specimen
substrate holder is a coverslip vise, referred to also as a
clip.
[0028] 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.
[0029] 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.
[0030] 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.
[0031] 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.
[0032] In at least one embodiment, the feeder reel and the take-up
reel are exposed.
[0033] 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.
[0034] In at least one embodiment, the reel-to-reel frame is
configured to integrate with an ultramicrotome knife-stage.
[0035] 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.
[0036] In one embodiment, the system places a cut resin section on
the polymer coverslip.
[0037] 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.
[0038] 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.
[0039] 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.
[0040] 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.
[0041] 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.
[0042] In another embodiment, a substrate positioned across the
surface block includes a coated Kapton.RTM. polyimide layer or
sheet.
[0043] In another embodiment, the substrate includes a carbon
coated polyimide substrate.
[0044] In another embodiment, the stub holder further includes a
graphene coated polyimide substrate.
[0045] 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.RTM.
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.
[0046] 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.RTM. polyimide
tape.
[0047] 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.
[0048] 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.
[0049] 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.
[0050] 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.
[0051] 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.
[0052] 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.
[0053] 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.RTM. 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.RTM. 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.RTM. polyimide tape from the feeder
reel to the take-up reel; positioning the coated Kapton.RTM.
polyimide tape between the edge of a 35.degree.-45.degree. angle,
2-8 mm diamond blade and water in the knife boat; receiving cut
resin sections from the water onto the coated Kapton.RTM. polyimide
tape; and advancing the coated Kapton.RTM. polyimide tape.
[0054] The method embodiments further include embodiments that
advance the coated Kapton.RTM. polyimide tape with respective
stepper motors that drive rotation of the feeder reel and the
take-up reel.
[0055] The method may further include embodiments that advance the
coated Kapton.RTM. polyimide tape in iterative steps.
[0056] The method may further include embodiments that advance the
coated Kapton.RTM. polyimide tape by continuously rotating the
feeder reel and the take-up reel.
[0057] The method may further include programming an electronic
control unit to advance the coated Kapton.RTM. polyimide tape.
[0058] The method may further include positioning the coated
Kapton.RTM. polyimide tape at the edge of a 35.degree.-45.degree.
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.
[0059] 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.RTM. polyimide tape.
[0060] The method may further include exposing the respectively cut
resin section to a chloroform vapor as the spreading agent.
[0061] The method may further include positioning an exhaust
conduit proximate the ultramicrotome to control a concentration of
chloroform vapor around the knife stage.
[0062] In yet another method of transferring samples from an
ultramicrotome to a coated Kapton.RTM. 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.RTM. polyimide tape
and/or a coated polymer coverslip to a knife-stage connected to the
ultramicrotome; positioning a coated Kapton.RTM. polyimide tape
and/or a coated polymer coverslip at the edge of a
35.degree.-45.degree. 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.RTM. polyimide
tape and/or a coated polymer coverslip.
[0063] 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.
[0064] 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.
[0065] 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.
[0066] The method of transferring samples may further include
connecting a coverslip vise for array tomography to the knife stage
adapter.
[0067] 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.
[0068] The method of transferring a cut resin section of a specimen
block attached to an ultramicrotome onto a coated Kapton.RTM.
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.
[0069] The method of transferring samples includes receiving a
thin, semi-thin, semi-thick or thick resin sections onto a coated
Kapton.RTM. polyimide tape.
[0070] The method of transferring samples includes using respective
motors to rotate the feeder reel and the take-up reel.
[0071] 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.
[0072] The method may further include storing the reel to reel
frame on a stand.
BRIEF DESCRIPTION OF THE DRAWINGS
[0073] 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.
[0074] FIG. 1 is a PRIOR ART side view of an ultramicrotome sample
preparation device positioned proximately to a knife stage
according to the background of this disclosure.
[0075] FIG. 2 is a side perspective view of an ultramicrotome
positioned proximately a knife stage to receive cut samples onto a
reel to reel tape according to embodiments of this disclosure.
[0076] FIG. 3A is a side perspective view of a knife stage adapter
as set forth according to embodiments of this disclosure.
[0077] FIG. 3B is a side view of a reel to reel assembly for
collecting cut resin samples from an ultramicrotome positioned
proximately to a knife stage according to this disclosure.
[0078] FIG. 3C is a side view of a reel to reel assembly for
collected cut resin samples from an ultramicrotome positioned
proximately to a knife stage according to this disclosure.
[0079] FIG. 4 is a disassembled view of a modular reel to reel
assembly for collecting cut resin samples from an ultramicrotome
positioned proximately to a knife stage according to this
disclosure.
[0080] FIG. 5 is a side view of a modular reel to reel assembly
connected to a knife stage adapter and configured for attaching to
a knife stage proximate an ultramicrotome for collecting cut resin
samples from the ultramicrotome positioned proximately to the knife
stage according to this disclosure.
[0081] FIG. 6 is a rear perspective view of a modular reel to reel
assembly for collecting cut resin samples from an ultramicrotome
positioned proximately to a knife stage according to this
disclosure.
[0082] FIG. 7 is a rear perspective view of a modular reel to reel
assembly for collecting cut resin samples from an ultramicrotome
positioned proximately to a knife stage according to this
disclosure.
[0083] FIG. 8 is a side perspective view of a modular reel to reel
assembly for collecting cut resin samples from an ultramicrotome
positioned proximately to a knife stage according to this
disclosure.
[0084] FIG. 9 is a top perspective view of a modular reel to reel
assembly for collecting cut resin samples from an ultramicrotome
positioned proximately to a knife stage according to this
disclosure.
[0085] FIG. 10 is a rear perspective view of a modular reel to reel
assembly for collecting cut resin samples from an ultramicrotome
positioned proximately to a knife stage according to this
disclosure.
[0086] FIG. 11 is a top side perspective view of a modular reel to
reel assembly for collecting cut resin samples from an
ultramicrotome positioned proximately to a knife stage according to
this disclosure.
[0087] FIG. 12A is a side plan view of a modular reel to reel
assembly for collecting cut resin samples from an ultramicrotome
positioned proximately to a knife stage according to this
disclosure.
[0088] FIG. 12B is a side plan view of a knife stage and associated
flanges and connectors for use with an ultramicrotome according to
this disclosure.
[0089] FIG. 12C is a side plan view of a knife stage and associated
flanges and connectors supporting a knife stage adapter connecting
a reel to reel assembly in functional proximity to an
ultramicrotome according to this disclosure.
[0090] FIG. 12D is a rear plan view of a knife stage configured for
use with a knife stage adapter according to embodiments of this
disclosure.
[0091] FIG. 13 is a top perspective view of a reel to reel frame
positioning a tape used to collect cut samples from an
ultramicrotome according to the embodiments of this disclosure.
[0092] FIG. 14 is a side perspective view of a reel to reel frame
positioning a tape used to collect cut samples from an
ultramicrotome according to the embodiments of this disclosure.
[0093] FIG. 15 is a side perspective view of a reel to reel frame
positioning a tape used to collect cut samples from an
ultramicrotome according to the embodiments of this disclosure.
[0094] FIG. 16 is a close-in side perspective view of a nose or
snorkel of a reel to reel frame positioning a tape in proximity to
an ultramicrotome to receive cut samples on the tape according to
the disclosure herein.
[0095] FIG. 17 is a close-in top perspective view of a nose or
snorkel of a reel to reel frame positioning a tape in proximity to
an ultramicrotome to receive cut samples on the tape according to
the disclosure herein.
[0096] FIG. 18 is a close-in top perspective view of a nose or
snorkel of a reel to reel frame positioning a tape in proximity to
an ultramicrotome to receive cut samples on the tape via a knife
boat according to the disclosure herein.
[0097] FIG. 19 is a rear top perspective view of reel motors
connected to a reel to reel frame according to embodiments of this
disclosure.
[0098] FIG. 20A is a side top perspective view of reel motors
connected to a reel to reel frame connected to a knife stage
adapter connected to a knife stage of an ultramicrotome according
to embodiments of this disclosure.
[0099] FIG. 20B is a side plan view of reel motors connected to a
reel to reel frame connected to a knife stage adapter connected to
a knife stage of an ultramicrotome according to embodiments of this
disclosure.
[0100] FIG. 21 is a rear perspective view of a feeder reel and a
take up reel assembly on a reel to reel frame connected to a knife
stage according to embodiments of this disclosure.
[0101] FIG. 22 is a top perspective view of a knife stage and
associated cartesian positioning control features for positioning a
knife boat relative to an ultramicrotome according to embodiments
of this disclosure.
[0102] FIG. 23 is a rear perspective view of a reel to reel frame
connected to a knife stage adapter with associated cartesian
positioning control features for positioning a knife boat relative
to an ultramicrotome according to embodiments of this
disclosure.
[0103] FIG. 24 is a top perspective view of a knife stage and
associated cartesian positioning control features configured for
modular removal relative to an ultramicrotome according to
embodiments of this disclosure.
[0104] FIG. 25 is a top perspective view of a knife stage and
associated cartesian positioning control features configured for
modular removal relative to an ultramicrotome according to
embodiments of this disclosure.
[0105] FIG. 26 is a top perspective view of a reel to reel frame
supporting a tape guide proximate a knife boat of a knife stage
according to aspects of this disclosure.
[0106] FIG. 27A is a top perspective view of a trigger holder for a
specimen that is adapted to connect to a knife stage adapter that
connects to a knife stage of an ultramicrotome according to this
disclosure.
[0107] FIG. 27B is a top perspective view of a trigger holder for a
specimen that is adapted to connect to a knife stage adapter that
connects to a knife stage of an ultramicrotome according to this
disclosure.
[0108] FIG. 28 is a side perspective view of a modular reel to reel
frame that has been disconnected from a knife stage adapter and
configured for storage on a stand according to embodiments
herein.
[0109] FIG. 29 is a side perspective view of a knife stage adapter
configured for receiving specimen holders according to the
embodiments herein.
[0110] FIG. 30 is a top perspective view of a central specimen
holder in the form of a vise that clips a substrate that receives a
cut specimen from a knife edge proximate an ultramicrotome
according to this disclosure.
[0111] FIG. 31 is a top perspective view of a central specimen
holder in the form of a vise that clips a substrate that receives a
cut specimen from a knife edge proximate an ultramicrotome
according to this disclosure.
[0112] FIG. 32A is a top perspective view of a central specimen
holder in the form of a vise that clips a substrate that receives a
cut specimen from a knife edge proximate an ultramicrotome
according to this disclosure.
[0113] FIG. 32B is a side perspective view of a central specimen
holder in the form of a vise that clips a substrate that receives a
cut specimen from a knife edge proximate an ultramicrotome
according to this disclosure.
[0114] FIG. 33 is a schematic view of an example computer
environment in which all electronic controls of this disclosure may
use with appropriate modification according to the embodiments
herein.
DETAILED DESCRIPTION
[0115] 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 [1/4''] wide.times.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.
[0116] 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.
[0117] By way of overview, this disclosure includes reference to
one non-limiting commercial embodiment of an ultramicrotome
illustrated in PRIOR ART FIG. 1. As shown therein, an
ultramicrotome is a sophisticated sampling and sectioning machine
that holds a sample (not shown in the figures, but may include
samples of biological tissue, tissue in paraffin blocks, cells
and/or tissue in resin blocks, as well as samples of inanimate
objects made of numerous materials to be analyzed). In a commonly
available microtome 150, the tissue samples or sample blocks are
held in a microtome sample attachment feature, such as the one
illustrated in FIG. 8, Ref. 814, and that sample attachment
feature, or nib 814, is an interchangeable part of an adjustable
and continuously operated ultramicrotome arm 29. The ultramicrotome
arm 152 may be configured to move up and down, pivoting along an
arc shaped groove or range of motion 27, to continuously shave
microscopically thin sample layers (or larger slices if desired)
for placement onto a slide or substrate for analysis under various
microscopy tools. An ultramicrotome 150, therefore, has associated
computerized components that control power, mechanical movement,
and general operation of the microtome system. Ultramicrotomes,
therefore, accommodate the collection of hundreds to thousands of
ultra-thin (thickness of 50 nm or less) and/or semi-thin (thickness
above 50 nm) sample sections or sample layers that can be
automatically and continuously collected from a diamond knife edge
onto a graphene-based, or other specialized material, surface
serving as a collection slide. These computer components are
accessible for user control via an ultramicrotome controller as
described herein.
[0118] Options in the ultramicrotome, such as the example prior art
of FIG. 1, allow the user to place appropriate knife assemblies
onto a knife stage. For example, and without limiting this
disclosure, setting up an ultramicrotome 150 as shown in FIG. 1
includes positioning a knife boat 16 onto a knife carrier 20. The
arcuately pivoting sample arm 29 moves in necessary dimensions 21
(up/down/arcuate), 23 (outward), 25 (inward) to continuously force
a sample, or specimen block, against a knife blade 14 to shave off
layers of ultra-thin and semi-thin sample sections of the overall
sample held in the microtome sample attachment feature, or nib 814.
The ultramicrotome has computer programmed memory to move the arm
in all dimensions to cut sample sections against a knife blade 14.
Prior embodiments of microtomes require retrieving the samples from
cooling water held in the open chamber of the knife boat 16 and
then further require placement of the sample sections on a slide
for viewing under a microscope. In some embodiments, the surface of
the distilled-deionized-filtered water contained in the knife boat
16 is maintained at the constant level during the sectioning
process by a programmable Aladdin Single-Syringe infusion pump 12
(e.g., 60 mL plastic syringe) thus, ensuring proper ultrathin
(thickness of 50 nm or less) and/or semi-thin (thickness above 50
nm) sectioning at the edge of the diamond knife. In some
embodiments, the knife boat has a triangular shaped bottom forming
the reservoir for water or other liquids.
[0119] 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
FIGS. 2, 3A, and 29 and includes a base portion 550B adapted to
connect to the knife stage 158 on an ultramicrotome 150. A platform
region 550C receives at least one adjustment screw 560 attaching
the platform region to the base portion 550B at a plurality of
selectable heights. An adapter edge 550A is configured to receive a
plurality of specimen substrate holders in a modular connection as
discussed herein. Accordingly, the knife stage adapter 550 is
configured to hold a number of different specimen holders,
including but not limited to, at least a reel to reel frame 182, a
trigger holder 300 (i.e., a scanning electron microscope stub
holder), a coverslip vise 435, and other mechanical devices that
can be positioned proximately to an ultramicrotome arm 152, a knife
edge (e.g., a diamond knife edge) 439, and a knife boat 800. In
some non-limiting embodiments, the diamond knife edge 439 may be
connected to the knife boat at a 35 degree to 45 degree angle and
may be approximately 2 mm to 8 mm wide. The knife stage adapter
places these different specimen holders in positions to receive
specimen slices onto a slide, a coverslip, or other specimen
substrates (including a coated Kapton.RTM. polyimide specimen tape)
that receives the specimens as slices or cut resin sections from a
specimen attachment feature (e.g., nib 814 of FIG. 16) via the
knife edge 439. The knife stage adapter 550 may include a lever arm
552 configured for arcuate movement relative to the base portion
550C and configured to adjust a position of the central specimen
substrate holder along the Z-axis as shown in FIG. 2B. Example
relationships and functional embodiments of
[0120] 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 FIGS. 3A-21, particularly FIGS. 4, 5, 8, 11
in conjunction with the knife stage adapter 550 attaching the
reel-to-reel frame to a knife stage 158 of an ultramicrotome 150.
The ultramicrotome may be any commercially available
ultramicrotome, and the reel-to-reel frame bolts into the knife
stage adaptor, which in turn bolts into the knife stage. In
particular, the knife stage adapter bolts into an upper section of
the knife stage. The knife stage 158 may be machined for precise
dimensions in suitable materials such as aluminum, carbon, copper,
chromium, brass, iron, molybdenum, nickel, stainless steel,
titanium alloys; typically these materials are susceptible to
extremely precise machining. As 3D printing moves forward, other
polymers may present equally accurate fabrication opportunities. In
one embodiment, the machined knife stage of FIGS. 3B and 3C is
modularly attached to a knife carrier 441 defining a receptacle 444
for holding a knife boat 800 in place as described herein. FIG. 3B
illustrates the flanges 164, 167 and support pillars 163A, 163B
holding the knife stage adaptor 185 to the knife stage 158, as
illustrated in FIGS. 12A-12D.
[0121] In one non-limiting embodiment of the reel to reel frame 182
shown in FIGS. 3A-3C, a feeder reel 175 is adapted to receive a
length of a specimen tape 190, wherein the feeder reel 175 is
rotatably coupled to the reel-to-reel frame 182. A take-up reel 177
is also adapted to receive the specimen tape 190 across the
reel-to-reel frame 182, and the reel-to-reel frame 182 is
configured to integrate with the knife stage adapter 550 and
connect to an ultramicrotome 150 that transfers cut samples onto
the specimen support tape 190. The specimen tape is not limited to
any one material but can include, without limitation, a graphene
coated tape, as described above, and the tape may also be a
conductive tape. These tapes hold resin based microscopic sections
of cells and tissues thereon and are sufficiently pliable to wind
around hubs of the feeder reel 175 and the take up reel 177 at a
desirable tension between the reels. In one embodiment, the
reel-to-reel frame 182 positions the feeder reel 175 and the
take-up reel 177 near the ultramicrotome with the reels being
exposed. It is notable that the reel-to-reel frame 182 holds the
feeder reel 175 and the take-up reel 177 such that the reels are
modular and separable components that connect to the reel to reel
frame 182. In this way, the reels can be transferred from specimen
collection operations of an ultramicrotome 150 to other machinery
such as numerous kinds of microscopes and imaging apparatuses.
[0122] 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.
[0123] 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 FIGS. 18, 24, and 25,
the reel to reel frame 182 is detachable from the knife stage
adapter 550 without removing the feeder reel 175 or the take-up
reel 177 or the specimen tape 190. The reel to reel frame 182 is
adapted to re-attach to additional imaging systems, and the
additional imaging systems are selected from but not limited to
secondary electron imaging systems, backscatter electron imaging
systems, and scanning transmission electron microscopy imaging
systems. The additional imaging systems are further selected from
quantitative measurement imaging systems comprising EDS and EELS
systems. FIGS. 4, 26 illustrate that the modular arrangement of
both the knife stage adapter 550 and the various central specimen
holders (i.e., a reel to reel frame, a trigger holder, and vise)
can be connected and removed numerous combinations to form a
plurality of assemblies. In FIGS. 4, 26, one non-limiting assembly
shows the reel to reel frame connected to the knife stage adapter
and that assembly is removably attached to the knife stage as
discussed herein. As shown in FIG. 28, components of this
disclosure may be stored on stand 193 for future use.
[0124] 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. FIG. 22
illustrates additional versatility by including numerous mechanisms
561 for adjusting a position of the knife stage adapter 550, a
reel-to-reel frame 182, a trigger holder 300, a clip holder or vise
435 (FIGS. 30, 31, 32A, 32B), and many other kinds of central
specimen holders. As shown in FIGS. 20-23, adjustment mechanisms
may be utilized with macro position adjustment knobs 560 and micro
position adjustment knobs 561 that can assist in moving the
apparatuses in the X, Y, and Z Cartesian coordinates of FIG. 2B.
These positional adjustments may also be implemented by many
different electronic and software driven machines as described
herein.
[0125] The versatility of these operations is shown in a system for
preparing cut samples with an ultramicrotome 150. In FIGS. 3-21,
the system is shown with a modular reel to reel assembly 185
connected to the knife stage adapter 550 and the ultramicrotome
150. In at least one embodiment, the system includes a reel to reel
frame 182 and a knife stage adapter 550, wherein the knife stage
adapter 185 is adapted to connect the modular reel to reel assembly
185 to the ultramicrotome 150. The modular reel to reel assembly
185 may be described as incorporating the reel to reel frame 182
along with components and combinations of components including, but
not limited to, a feeder reel 175 connected to a length of a
specimen tape 190, wherein the feeder reel 175 is rotatably coupled
to the reel-to-reel frame 182. A take-up reel 177 is connected to
the reel to reel frame 182 and receives the specimen support tape
190 across the reel-to-reel frame 182. In one non-limiting
embodiment, the reels are removable from the reel to reel frame
with or without disassembling the knife stage 158, the knife stage
adapter 550, or the specimen tape 190. The reels 175, 177 may be
attached to a separate plate that connects by various fasteners to
the reel to reel frame 182.
[0126] As shown in FIGS. 10, 19 and others, the reel to reel frame
182 connects to or supports, a feeder motor 725B that drives
rotation of the feeder reel 175 and a take-up motor 725A that
drives rotation of the take-up reel 177. It is important to note
that in some embodiments, the reel to reel assembly 185 includes
only the feeder reel 175 and the take up reel 177 that are
separable from the reel-to-reel frame 182. In other embodiments,
the system may include a plurality of sample position sensors 1410,
1420 connected to the modular reel to reel assembly 185 and/or the
ultramicrotome 150 that sends data to the electronic controller 700
(FIGS. 7, 9) regarding where the ultramicrotome specimen cantilever
arm 152 is located in its arcuate path. Other embodiments utilize a
single sensor on the platform of the ultramicrotome to detect where
the ultramicrotome specimen cantilever arm 152 is located on its
path. In this way, the electronic controller described below is
updated with the position of the sample on the ultramicrotome
specimen cantilever arm 152 and knows when the specimen tape 190
must advance to maintain continuity. The electronics and
programming of the electronic controller 700 are discussed in more
detail below. The positions of the sensors 1410, 1420 are not
limited to any one installation and can be relocated for the use at
hand. With data from the sensors 1410, 1420, at least one
electronic control unit 700 is equipped for controlling respective
speeds of the feeder motor 725B and the take-up motor 725A, wherein
the electronic control unit 700 adjusts the respective speeds
according to a sample position signal received from the plurality
of sample position sensors 1410, 1420. As shown in the figures, the
ultramicrotome specimen cantilever arm connects to an
ultramicrotome chuck that has the nib 814 for holding a specimen
block to be cut.
[0127] The system of FIGS. 3-21 further illustrates an
ultramicrotome specimen cantilever arm 152 holding a specimen block
in engagement with a knife edge 439 connected to a knife boat 800,
wherein the sample position sensors include at least the first
sensor 1410 on the knife boat and a second sensor 1420 on the
ultramicrotome specimen cantilever arm 152, but these positions are
entirely optional. The specimen tape 190 is positioned to receive
samples on the specimen tape 190 from the knife edge 439 attached
to the knife boat 800 connected to a knife carrier 20 connected to
the ultramicrotome 150. The specimen tape traverses the tape guide
180 across a nose or snorkel 438 as shown in FIG. 20 (i.e., a
portion of the tape guide that is submerged into water in the knife
boat 800 filled from pump line 1050 (FIG. 10) to pump 1060 (FIG.
10). In one embodiment, the tape guide 180 may include a joint 181
that allows for different heights of the snorkel or nose end 438.
The tape guide, therefore, may have multiple degrees of freedom in
the x, y, and z directions for the best placement of the tape in
receiving a specimen slice. As shown in FIGS. 13-15 and FIG. 17,
the nose end 438 of the tape guide is moveable by the various
position controllers of the knife stage, the knife stage adapter,
and the reel to reel frame. The nose end 438 is positioned relative
to a knife edge 439 to receive a cut specimen onto a tape 190.
[0128] As noted above in regard to FIGS. 7 and 9, operation of the
system may be coordinated by an electronic controller 700 that may
further include a graphical user interface 154 connected to the
electronic control unit 700 and configured to receive data entry
for programming the electronic control unit. The data entry may
include programming optional parameters to maintain variables such
as speed selections for the feeder motor 725B and the take-up motor
725A, which in one embodiment may be a stepper motor. The
electronic controller 700 may be characterized as having general
purpose inputs and outputs that are connected to appropriate
processors, computerized memory, and hardware that is appropriate
for customized machine logical operations. In one non-limiting
example, the electronic controller receives data from a system of
sensors, including a first cantilever arm position sensor and a
second cantilever arm position sensor, either of which may be
located on the cantilever arm and/or the body of the
ultramicrotome. This data, along with speed data for the motors can
be used for numerous control systems programmed into the electronic
controller. For example, in one embodiment, by using the speed of
the motors and the rotation speed of the reels, along with
dimensions of the tape and linear speed of the tape, the electronic
controller is adapted to track position coordinates of each cut
resin section that is located on the tape. In this way, the
electronic controller includes a computer registry or database that
allows for tracking where a specimen is located along the tape and
which sections of the tape are empty. This is useful information
when the tape with specimens thereon are "played back" for
microscopy purposes.
[0129] This disclosure accounts for multiple, non-limiting versions
of the firmware for the control environment, illustrated in FIGS. 7
and 33 at least. Each version consists of several modules for
different tasks. In one embodiment, software for the electronic
controller 700 includes a tape control module. Its task is to
maintain a constant speed of the tape 190 by taking the two spool
diameters (i.e., the diameters of hubs 173A, 173B) and spool
revolutions per unit time into consideration and using this
information to control the two respective step motor speeds,
connected to the spools. Another feature of the module is the
access of the hardware settings of the motor controller on the
board. The communication module uses a transmission protocol to
communicate with a user interface software via the USB interface of
the hardware.
[0130] 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.
[0131] 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.
[0132] 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.
[0133] 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.
[0134] 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.
[0135] 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.
[0136] The figures, such as but not limited to FIGS. 6, 7, and 8,
further illustrate that the system may include specimen unfolding
additives to the environment. When the sample is sliced from a
sample block attached to a nib 814 on the ultramicrotome arm 152,
its initial contact with the knife edge 439 moves the sample slice
into water of the knife boat 800, and that process tends to fold
the sample or crimple the surface of the sample. Systems of this
disclosure utilize additives to the environment of the knife boat
800 to unfold the sample. In one non-limiting embodiment, the
unfolding agent is chloroform 614 as shown in the figures emitted
from a mister assembly that directs the chloroform toward the
sliced sample to unfold it. As shown in FIG. 6, the system also
includes an exhaust arrangement 625 to remove excess gaseous
additives such as chloroform from the work area.
[0137] 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 FIGS.
30-32, a clip, or vise, 435 is attached to the knife stage adapter
550, wherein the clip or vise 435 is configured to hold a graphene
substrate in a position to receive the cut sample thereon. A lever
arm 552 is configured for arcuate movement about the knife stage
adapter 550 to position a nose end 437 of the clip 435 at
selectable points along the Z axis and adjacent a sample block on
the ultramicrotome 150.
[0138] A sample substrate holder may also encompass a trigger
holder as illustrated in FIGS. 27A, 28B for array tomography and
possibly for additional use with scanning electron microscopes. The
trigger holder body 300 is illustrated extensively in FIGS. 27A and
27B for use with an ultramicrotome. As illustrated in FIGS. 27A,
27B, the substrate holder body 300 defines a passageway with a
first opening 340 at one end of the holder body 300 and a second
opening 335 extending from the first opening 340 toward a second
end of the holder body. A trigger 325 fits within the second
opening 335, wherein the trigger 325 is actuated by a spring 330
connected to the trigger 325 and the holder body 300. A removable
surface block 310 is configured to fit within the first opening 340
of the holder body 300, and the removable surface block 310 is held
in place by the trigger 325 biased by the spring 330. In operation,
the knife stage adapter 550 positions the removable surface block
310 proximate the ultramicrotome 150. A sample can be collected
onto a graphene sample substrate that is positioned across the
surface block 310 and held in place between sides 312A, 312B of the
surface block 310 connected to respective sections 339A, 339B of
the holder body 330 and the trigger 325.
[0139] 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 (FIG. 16) of the ultramicrotome 150;
connecting a length of the specimen tape 190 between a feeder reel
175 and a take-up reel 177 connected to a reel to reel frame 182;
advancing the specimen tape 190 from the feeder reel 175 to the
take-up reel 177; positioning the specimen tape 190 between the
sample block and a knife stage 158 connected to the ultramicrotome
150, wherein a knife edge 439 mounted on the knife stage 158
receives a portion of the sample block against a knife edge 439 to
slice at least one sample of the sample block; and receiving the
sample on the specimen support tape 190 and advancing the specimen
support tape. The method continues by advancing the specimen
support tape 190 with respective stepper motors 1410, 1420 that
drive rotation of the feeder reel and the take-up reel. Advancing
the specimen support tape 190 may occur in iterative steps or on a
continuously driven tape. 26. The method of any one of Claims
23-25, further comprising programming an electronic control unit
700 to advance the specimen support tape 190.
[0140] 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.
[0141] 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.
[0142] 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.
[0143] 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.
[0144] 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.
[0145] 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.
[0146] 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.
[0147] The figures utilize an exemplary computing environment in
which example embodiments and aspects may be implemented. The
computing device environment of FIG. 33 is only one example of a
suitable computing environment and is not intended to suggest any
limitation as to the scope of use or functionality.
[0148] 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.
[0149] 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.
[0150] 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.
[0151] 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 FIG. 2 by removable storage and
non-removable storage.
[0152] 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.
[0153] 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.
[0154] 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.
[0155] 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.
* * * * *