U.S. patent application number 11/759355 was filed with the patent office on 2007-10-04 for integrated crystal mounting and alignment system for high-throughput biological crystallography.
Invention is credited to Carl W. Cork, Earl W. Cornell, Thomas N. Earnest, Joseph M. Jaklevic, William Kolbe, Robert A. Nordmeyer, Bernard D. Santarsiero, Gyorgy P. Snell, Raymond C. Stevens, Derek Yegian.
Application Number | 20070228049 11/759355 |
Document ID | / |
Family ID | 23335932 |
Filed Date | 2007-10-04 |
United States Patent
Application |
20070228049 |
Kind Code |
A1 |
Nordmeyer; Robert A. ; et
al. |
October 4, 2007 |
INTEGRATED CRYSTAL MOUNTING AND ALIGNMENT SYSTEM FOR
HIGH-THROUGHPUT BIOLOGICAL CRYSTALLOGRAPHY
Abstract
A method and apparatus for the transportation, remote and
unattended mounting, and visual alignment and monitoring of protein
crystals for synchrotron generated x-ray diffraction analysis. The
protein samples are maintained at liquid nitrogen temperatures at
all times: during shipment, before mounting, mounting, alignment,
data acquisition and following removal. The samples must
additionally be stably aligned to within a few microns at a point
in space. The ability to accurately perform these tasks remotely
and automatically leads to a significant increase in sample
throughput and reliability for high-volume protein characterization
efforts. Since the protein samples are placed in a
shipping-compatible layered stack of sample cassettes each holding
many samples, a large number of samples can be shipped in a single
cryogenic shipping container.
Inventors: |
Nordmeyer; Robert A.;
(US) ; Snell; Gyorgy P.; (US) ; Cornell;
Earl W.; (US) ; Kolbe; William; (US) ;
Yegian; Derek; (US) ; Earnest; Thomas N.;
(US) ; Jaklevic; Joseph M.; (US) ; Cork;
Carl W.; (US) ; Santarsiero; Bernard D.;
(US) ; Stevens; Raymond C.; (US) |
Correspondence
Address: |
LAWRENCE BERKELEY NATIONAL LABORATORY
ONE CYCLOTRON ROAD, MAIL STOP 90B
UNIVERSITY OF CALIFORNIA
BERKELEY
CA
94720
US
|
Family ID: |
23335932 |
Appl. No.: |
11/759355 |
Filed: |
June 7, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11064357 |
Feb 22, 2005 |
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11759355 |
Jun 7, 2007 |
|
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|
10319282 |
Dec 12, 2002 |
6918698 |
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11064357 |
Feb 22, 2005 |
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60341020 |
Dec 12, 2001 |
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Current U.S.
Class: |
220/560.07 ;
378/73; 62/64 |
Current CPC
Class: |
B01L 2200/18 20130101;
G01N 23/207 20130101; B01L 2300/1883 20130101; B01L 9/065 20130101;
C30B 35/005 20130101; C30B 33/00 20130101; C30B 29/58 20130101;
G01N 1/42 20130101; G01N 23/20025 20130101 |
Class at
Publication: |
220/560.07 ;
378/073; 062/064 |
International
Class: |
F17C 3/00 20060101
F17C003/00; F25D 17/02 20060101 F25D017/02; G01N 23/207 20060101
G01N023/207 |
Goverment Interests
STATEMENT REGARDING FEDERAL FUNDING
[0002] This invention was made with U.S. Government support under
Contract Number DE-AC03-76SF00098 between the U.S. Department of
Energy and The Regents of the University of California for the
management and operation of the Lawrence Berkeley National
Laboratory. The U.S. Government has certain rights in this
invention.
Claims
1. A sample assembly comprising: a. a sample base, b. a sample
tube, wherein one of two ends of the sample tube is affixed to the
sample base; c. a sample loop affixed to the other end of the
sample tube, wherein d. a sample is affixed by the sample loop.
2. The sample assembly of claim 1 wherein said sample base is
ferromagnetic.
3. The sample assembly of claim 2 wherein said sample base is
essentially axisymmetric.
4. An apparatus for transporting cryogenically frozen biological
crystal samples with the sample assembly of claim 1, comprising: a.
a cryogenic transport container, and b. a cassette carrier with a
plurality of shelves retained by a sheet, the cassette carrier
removably securing a plurality of sample cassettes within the
cassette carrier unit, with each of the sample cassettes
independently secured, wherein the cassette carrier is packaged in
the cryogenic transport container for shipping at cryogenic
temperatures to transport a plurality of cryogenically frozen
biological crystal samples with the sample assembly of claim 1
5. The apparatus for transporting cryogenically frozen biological
crystal samples of claim 4 wherein the cassette carrier comprises:
one or more of the sample assemblies of claim 1.
6. The apparatus for transporting cryogenically frozen biological
crystal samples of claim 4 wherein the sample cassette further
comprises: a. a cassette base with at least one through opening for
loosely receiving a sample assembly; b. a magnet inserted into a
recess in the cassette base, the magnet containing an opening
aligned with, and opened to the corresponding cassette base through
opening; c. at least one keyed shaft mounted in the cassette base;
wherein said cassette base can loosely accept one of the sample
assemblies in each cassette base through opening and is removably
retained by said magnet; and d. a cassette cover with 1. a keyed
opening to accept the cassette base keyed shaft to substantially
prevent rotation, and 2. a recess corresponding to each cassette
base through opening, wherein each of said recesses positively
retains the corresponding sample assembly and protects the sample
attached to the sample assembly.
7. The cassette base of claim 6 wherein said cassette base is
ferromagnetic.
8. The cassette base of claim 7 wherein said magnet is further
comprised of a flexible machinable magnetic material.
9. A sample gripper comprising: a. a grasping means for retractably
capturing one of the sample assemblies of claim 1, b. a cooling
means for drawing liquid nitrogen up and around the captured sample
assembly, and c. a flowing means for sheathing the captured sample
assembly from direct contact with ambient moist air.
10. A sample gripper capable of gripping and moving one of the
sample assemblies of claim 1, comprising: a. a retractable split
collet with a collet actuation surface, a collet interior, and a
collet recess; b. an inner tube, mounted to a gripper flange, which
compresses the collet actuation surface upon retraction of the
split collet, causing the collet recess to contract upon the sample
assembly of claim 1, thus forcibly retaining the sample assembly;
c. a shroud tube of larger diameter than the inner tube, forming an
outer shroud area between the inner tube and the shroud tube; and
d. a plurality of small openings in the gripper flange connecting
to a port, communicating a flow from the port to the inner shroud
area; wherein a warm dry gas can be applied to the port to keep
frost from forming on the inner tube and the split collet, and a
gage vacuum can be applied to the port to force liquid nitrogen
into the collet interior when the outer shroud area is immersed in
liquid nitrogen.
11. A sample repository comprising: a. a storage Dewar, with an
external referencing feature substantially immovably attached to
said Dewar; b. an internal referencing feature substantially
immovably attached to the storage Dewar; c. wherein said internal
referencing feature is substantially aligned with said external
referencing feature such that a translation and rotation of said
external referencing feature produces in the internal referencing
feature a known movement substantially the same as said translation
and rotation.
12. A sample assembly comprising: a. a sample base, b. retaining
means, whereby a sample is affixed to the sample base.
13. The sample assembly of claim 1 wherein said retraining means
comprises: a. a loop of material retained by a tube affixed in the
sample base; b. wherein the loop of material retains the
sample.
14. The sample assembly of claim 12 wherein said sample base is
essentially axisymmetric.
15. An apparatus for transporting cryogenically frozen biological
crystal samples on the sample assemblies of claim 12, comprising:
a. a cryogenic transport container, and b. a transport means
removably inserted into the cryogenic transport container, whereby
a plurality of the sample assemblies of claim 12 may be transported
in a cryogenic state.
16. The apparatus for transporting cryogenically frozen biological
crystal samples of claim 15 wherein the transport means further
comprises: a. a cassette base with at least one through opening for
loosely receiving one or more of the sample assemblies of claim 12;
b. a magnet inserted into a recess in the cassette base, the magnet
containing an opening aligned with, and opened to the corresponding
cassette base through opening; c. at least one keyed shaft mounted
in the cassette base; wherein said cassette base can loosely accept
one of the sample assemblies in each cassette base through opening
and is removably retained by said magnet; and d. a cassette cover
with 1. a keyed opening to accept the cassette base keyed shaft to
substantially prevent rotation, and 2. a recess corresponding to
each cassette base through opening, wherein each of said recesses
positively retains the corresponding sample assembly and protects
the sample attached to the sample assembly.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This divisional patent application claims benefit of
divisional U.S. patent application Ser. No. 11/064,357 filed on
Feb. 22, 2005, and entitled "Integrated Crystal Mounting and
Alignment System for High-throughput Biological Crystallography",
hereby incorporated by reference in its entirety, which in turn
claims priority to divisional U.S. patent application Ser. No.
10/319,282 filed on Dec. 12, 2002, and entitled "Integrated Crystal
Mounting and Alignment System for High-throughput Biological
Crystallography", issued as U.S. Pat. No. 6,9180,698 on Jul. 19,
2005, hereby incorporated by reference in its entirety, which in
turn claims priority to Provisional Patent Application No.
60/341,020, filed on Dec. 12, 2001, and also entitled "Integrated
Crystal Mounting and Alignment System for High-throughput
Biological Crystallography", which is hereby incorporated by
reference in its entirety.
REFERENCE TO A COMPUTER PROGRAM
[0003] Not applicable.
BACKGROUND OF THE INVENTION
[0004] 1. Field of the Invention
[0005] The present invention generally relates to the
transportation, robotic crystal mounting and alignment,
manipulation, mounting, alignment of crystal samples in a variety
of experimental environments. The present invention more
particularly relates to the mounting, alignment, and exposure of
samples to synchrotron radiation for high-speed x-ray
crystallography.
[0006] 2. Description of the Relevant Art
Overview of X-Ray Crystallographic Systems
[0007] Aspects of the present invention facilitate the
transportation, as well as the remote and unattended mounting and
alignment of frozen crystals--of e.g. biological materials, such as
proteins, lipids, or deoxyribonucleic acids (DNA)--for x-ray
diffraction analysis. A major challenge in the x-ray diffraction
analysis system design is the necessity of storing the samples in
liquid nitrogen before mounting and following removal, as well as
maintaining the samples at near liquid nitrogen temperature
throughout the mounting, alignment, and x-ray diffraction analysis
data acquisition process. Additionally, the precision and stability
of the crystal sample location alignment must be very high, with
absolute sample position maintained within a few microns while
rotating in one axis while being exposed to an incident x-ray beam
through as much as a full 360.degree..
[0008] Traditional x-ray diffraction analysis crystal sample
handling procedures are operator-intensive, requiring continuous
manual operator intervention at the measurement station. The
ability to perform these tasks remotely and automatically
significantly increases crystal mounting and measurement
throughput, as well as reliability for large-scale protein
crystallography characterization. An increase in throughput
multiplies the number of samples that may be analyzed in a given
time period, thus decreasing the time per sample, thereby lowering
the cost associated with synchrotron-based x-ray
crystallography.
Synchrotron-Based X-Ray Crystallography
[0009] One embodiment of this invention is in the area of cryogenic
protein crystallography at synchrotron sources, although the
robotic mounting and alignment system can be adapted for other
laboratory x-ray sources. Potential uses include high-volume
protein characterization experiments. The level of application of
this invention could range from a small experimental program
processing only a few samples per day to large projects screening
and analyzing many thousands of samples per year.
[0010] Synchrotron-based x-ray crystallography is one application
of this invention. Synchrotrons are capable of producing intense
monochromatic pseudo-coherent photons of precisely controllable
energies. The property of high intensity (otherwise known as high
brightness) of the synchrotron x-ray beams means that acquisition
of crystal lattice diffraction patterns can be done very rapidly,
whereas other, lower intensity beams may require several times
longer for a diffraction pattern to be acquired. The high
brightness of the synchrotron radiation, combined with the narrow
energy bandwidth achievable using a monochromator, can lead to
exceedingly high-resolution x-ray diffraction patterns.
[0011] The x-ray diffraction patterns can subsequently be analyzed
to infer the relative spatial positions of the atoms constituting
the crystal lattice structure. The overall x-ray diffraction
analysis of crystals is known as x-ray crystallography. The
information contained in the crystal structure can lead to
important insights about the function of the molecule and into
molecular-chemical interactions. Such insights can lead to
targeted, and thus faster, pharmaceutical development and improved
pharmaceuticals: a field known as `structure based pharmaceutical
design`.
Biological Crystallography Mounting Techniques
[0012] Currently, most x-ray crystallography work is done using
synchrotron x-ray sources. These x-ray sources are extremely
expensive to operate, which means that time is precious. However,
since synchrotron x-ray crystallography is still a recent
phenomenon, most sample mounting is done manually, which is both
slow and imprecise. Furthermore, since crystallography must be done
on crystalline material, the sample must be maintained in a frozen
state. Typically, this is ensured by keeping the sample at near
liquid nitrogen temperatures.
[0013] The requirement that the sample be maintained at liquid
nitrogen temperature, however, requires that technicians and
scientists can only mount the samples using cumbersome techniques
of indirectly handling the sample. Thus, people cannot be allowed
to inadvertently heat the sample, and reciprocally, the sample
handling tools, and sample handling fixtures, cannot freeze the
fingers of the people who do the mounting. To meet this
requirement, clumsy tools resembling forceps or pliers are used.
These tools are somewhat cumbersome, further adding time and
difficulty in mounting and handling the sample.
[0014] As more time is required to manually mount the sample, more
heat is transferred from the ambient atmosphere, raising the
crystal sample temperature. Some biological crystal samples, frozen
at a critical point in a chemical reaction with another compound,
continue their reactions at temperatures as low at 100.degree. K,
only about 22.degree. K above that of liquid nitrogen. This
stringent maximum temperature requirement for some samples implies
that the sample must be actively cooled during the entire mounting
process, which adds still further time and complexity to the
mounting process. It is preferable that the sample crystals be
cooled to a temperature not in excess of 150.degree. K, more
preferably not in excess of 130.degree. K, yet more preferably not
in excess of 110.degree. K, still more preferably not in excess of
100.degree. K, yet still more preferably not in excess of
90.degree. K, and most preferably not in excess of 80.degree.
K.
[0015] The largest time-related issue with manual operator mounting
of synchrotron x-ray crystallography samples is that the humans
must enter the x-ray irradiation area (`the hutch`) to mount and
dismount the crystal samples. This action involves in turn a
sequence of safety interlocking steps to protect the personnel from
a harmful and potentially lethal dosage of x-rays used to irradiate
the crystal sample to generate the diffraction patterns. Typically,
one or more heavy lead-lined doors must be opened and closed,
additional beam shutters inserted, and interlocking safety devices
must be carefully verified for safe operation, prior to human
access to the sample.
[0016] The result is that manual mounting of a synchrotron x-ray
crystallography sample is slow. As a result of being slow, manual
mounting is very expensive as measured in synchrotron beam
time.
Biological Crystallography Sample Transportation
[0017] Currently, there are relatively few synchrotron x-ray
sources available for x-ray crystallography. Therefore, scientists
wishing to use synchrotron x-ray sources face the dilemma of
transporting the crystal samples to the synchrotron while
simultaneously maintaining the crystal's cryogenically frozen
state.
[0018] A particularly fruitful use of synchrotron x-ray
crystallography is in detection of chemical interactions within a
specific biological sample. These interactions are evanescent in
nature, sometimes reacting in the one-nanosecond time scale.
Additionally, typical biological processes can follow a number of
biochemical pathways that are time dependent. Some of these
biochemical pathways proceed even at temperatures as low as
100.degree. K. Thus, for a scientist to determine the crystalline
structure of an intermediate state biochemical interaction, the
biological sample must be frozen to a temperature low enough to
inhibit further reaction, typically close to the liquid nitrogen
temperature of about 77.degree. K under normal laboratory
conditions.
A Relevant Patent
[0019] Abbott Laboratories is the named assignee of U.S. Pat. No.
6,404,849 B1 (the '849 patent), entitled "Automated Sample Handling
for X-Ray Crystallography". The '849 patent discloses algorithms
for centering a crystal at a reference position relative to home
position sensors, as well as the hardware for screwing a threaded
sample holding device on and off a positioning device. The '849
patent uses a multi-axis robot to move crystals from a sample rack
to a positioning device.
SUMMARY OF THE INVENTION
[0020] One aspect of the present invention is directed toward the
transportation and manipulation of samples of cryogenically frozen
biological particles, preferably protein crystals, mounted on
standardized base-pin configured sample assemblies.
[0021] The integrated crystal mounting and alignment system for
high-throughput biological crystallography which transports and
manipulates the sample assemblies comprises eight major components:
[0022] 1) a sample repository having a storage Dewar filled with
liquid nitrogen, capable of keeping many samples cryogenically
frozen, with a sample repository stage able to addressably move the
sample assemblies to a point where a particular sample assembly can
be extracted; [0023] 2) a system for shipping, storing and handling
of the sample assemblies at cryogenic temperatures, preferably
liquid nitrogen temperatures; [0024] 3) a computer-controlled
sampling system sequencing a particular sample assembly through the
steps of: a) selecting the particular sample assembly from the
cryogenic sample repository, b) removing the selected sample
assembly from the sample repository, c) transferring the sample
assembly to a three axis positioner mounted on a goniometer head,
d) centering the sample in the x-ray beam, e) exposing the sample
(held by the mounted sample assembly) to x-ray radiation to produce
a crystallographic image at a sequence of rotational exposure
angles while simultaneously maintaining the sample's cryogenic
temperatures, and f) replacing the sample assembly back in the
cryogenic sample repository; [0025] 4) a sample gripper capable of
firmly grasping a sample assembly, while keeping the sample at
cryogenic temperature; [0026] 5) a gripper stage, using the sample
gripper to: remove the sample assembly from cryogenic sample
repository, transport the sample to a sample positioner, and
replace the sample assembly in the sample repository, while at all
times maintaining the temperature of the sample at or below
78.degree. K; [0027] 6) a sample gripper defroster capable of
keeping the sample gripper free of frost buildup during cycles of
sample assembly mounting and dismounting (or unmounting) in ambient
humid air; [0028] 7) a sample positioner consisting of a precision
three-axis positioner mounted on a precision goniometer; and [0029]
8) an optical alignment system that provides feedback to the sample
positioner for precise alignment of the sample to a predefined
point in space within the x-ray beam during sample rotation.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] FIG. 1 is a cross sectional view of a sample assembly with a
crystal sample mounted.
[0031] FIG. 2A is a top view of a sample cassette with one sample
assembly.
[0032] FIG. 2B is a cross sectional view through section 2-2 of
FIG. 2A of a sample cassette with one sample assembly.
[0033] FIG. 2C is an exploded view of the cross sectional view of
FIG. 2B, with a sample cassette with one sample assembly.
[0034] FIG. 3A is a top view of a sample cassette cover.
[0035] FIG. 3B is a cross sectional view through section 3-3 of
FIG. 3A of a sample cassette cover.
[0036] FIG. 3C is a bottom view of a sample cassette cover showing
liquid nitrogen venting features.
[0037] FIG. 4A is a cross sectional view of a sample cassette
assembly comprised of an assembled sample cassette with one sample
assembly, and protected by a sample cassette cover.
[0038] FIG. 4B is an exploded cross sectional view of a sample
cassette assembly comprised of a sample cassette, one sample
assembly, and a sample cassette cover, before assembly.
[0039] FIG. 5A is a cross sectional view of a sample cassette
carrier with six assembled sample cassettes present and the top
slot vacant.
[0040] FIG. 5B is a bottom view of the sample cassette carrier and
all assembled sample cassettes absent.
[0041] FIG. 6 is a cross sectional view of a cryogenic shipping
container with sample cassette carrier with six sample cassette
assemblies present, and the top slot vacant.
[0042] FIG. 7A is a top view of a cassette deck with three sample
cassettes present and one sample cassette absent.
[0043] FIG. 7B is a sectional view of the cassette deck of FIG. 7A
with three sample cassettes present and one sample cassette
absent.
[0044] FIG. 8 is a cross sectional view of a sample gripper.
[0045] FIG. 9A is a partial front view of the integrated robotic
crystal mounting and alignment system showing most of the major
subsystems, where the system has just grasped a sample assembly in
the sample repository.
[0046] FIG. 9B is a partial front view of the integrated robotic
crystal mounting and alignment system showing most of the major
subsystems, where the system has retracted a pneumatic stage, known
as SmallMove, causing the sample gripper to move vertically
upwards.
[0047] FIG. 9C is a partial front view of the integrated robotic
crystal mounting and alignment system showing most of the major
subsystems, where the system has retracted the transverse vertical
stage, known as UpDown, causing the sample gripper to move further
vertically upwards.
[0048] FIG. 9D is a partial front view of the integrated robotic
crystal mounting and alignment system showing most of the major
subsystems, where the system has rotated the pneumatic 90.degree.
rotary stage, known as Rotary, causing the sample gripper to rotate
toward the sample positioner.
[0049] FIG. 9E is a partial front view of the integrated robotic
crystal mounting and alignment system showing most of the major
subsystems, where the horizontal stage has moved the sample gripper
in a long horizontal translation, causing the sample gripper to
move to a predetermined distance toward the sample positioner.
[0050] FIG. 9F is a partial front view of the integrated robotic
crystal mounting and alignment system showing most of the major
subsystems, where the SmallMove stage has moved the sample gripper
a short horizontal translation to place the sample assembly in
contact with the mounting post of the sample positioner.
[0051] FIG. 9G is a partial front view of the integrated robotic
crystal mounting and alignment system showing most of the major
subsystems, where the horizontal stage has moved the sample gripper
in a long horizontal translation, causing the sample gripper to
move to a predetermined distance away from the sample positioner,
leaving a sample assembly on the sample positioner mounting
post.
[0052] FIG. 10A is a partial front view of the sample positioner,
including a tilt plate disposed between a goniometer and the X' Y'
compound stage.
[0053] FIG. 10B is a partial front view of the sample positioner,
including another tilt plate disposed between the X' Y' compound
stage and the Z' stage.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
Definitions
[0054] "Biological crystal" means a crystallized frozen biological
material, preferably a cryogenically frozen biological material at
near liquid nitrogen temperatures.
[0055] "Biological material" means either a collection of
independent molecules, or a material having a non-covalently bound
assembly of molecules derived from a living source. Examples
include, but are not limited to, complexes of proteins, lipoprotein
particles comprised of lipoproteins and lipids; viral particles
assembled from coat proteins and glycoproteins; immune complexes
assembled from antibodies and their cognate antigens,
deoxyribonucleic acids (DNA), ribonucleic acids (RNA),
polysaccharides, etc.
[0056] "Computer" means any device capable of performing the steps
developed in this invention to result in an optimal waterflood
injection, including but not limited to: a microprocessor, a
digital state machine, a field programmable gate array (FGPA), a
digital signal processor, a collocated integrated memory system
with microprocessor and analog or digital output device, a
distributed memory system with microprocessor and analog or digital
output device connected with digital or analog signal
protocols.
[0057] "Cryogenic" means a temperature at or below that of liquid
nitrogen at standard atmospheric pressure, -195.79.degree. C. or
77.36.degree. K.
[0058] "Degree of freedom" means any one of the ways that a
mechanical system can change spatial configuration, examples
include, but are not limited to rotation, translation, and
combinations of rotations and/or translations in one or more
axes.
[0059] "Gage vacuum" means a relative pressure lower than ambient
atmospheric pressure.
[0060] "Ferromagnetic" means a material capable of exhibiting
alignment of atomic or molecular magnetic domains. Such a material
is capable of magnetization, and is subjected to forces in the
presence of a magnetic field.
[0061] "Goniometer" typically means a device for measuring angles.
Herein, it is used somewhat differently in that it rotates a
surface to particular angles as instructed by a computer device
using internal reference angular measurements.
[0062] "Dewar" means any container for liquefied gases. These
containers are traditionally typically double-walled with an
evacuated interior having low thermal emissivity surfaces to reduce
heat transfer from the container interior to the ambient
atmosphere.
Overview
[0063] The present invention is directed toward the transportation
and manipulation of samples of cryogenically frozen biological
materials, preferably protein crystals mounted on sample assemblies
comprised of a standardized base-pin configuration.
[0064] When the hardware components are computer operated as a
fully integrated crystal mounting and alignment system for
high-throughput biological crystallography (herein referred to as
the "system"), remote sample mounting and alignment of individual
samples to a predetermined position in space (for subsequent X-ray
illumination) can be achieved in less than 30 seconds with minimal
remote operator involvement. In some instances, samples have been
remotely mounted and aligned in a cycle of 20 seconds. Since no
human operator physical presence is required in the room where
active x-ray irradiation is taking place, there is no prolonged
sequence of doors or interlocks to safely admit an operator,
further increasing the speed of sample cycling.
[0065] The sample assembly used in this invention is comprised of a
sample base to which a thin sample tube is attached. The end of the
sample tube not attached to the sample base has a sample loop,
which contains the sample of frozen biological material to be
examined. Typical samples range from 5-200 .mu.m in size. The pin
has a loop, which contains a cryogenically frozen sample.
[0066] The system for shipping, storing and handling the samples is
comprised of a standardized sample cassette containing positions
for a plurality of sample assemblies, preferably 16 sample
assemblies each in the present design. The outer dimension
(diameter) of these sample cassettes is constrained by the maximum
inner diameter of a standard cryogenic transport container.
Presently, a preferred standard cryogenic transport container will
accommodate seven of these disks representing a maximum of 112
sample assemblies. Improvements on this design would allow even
more sample assemblies in a higher packing density arrangement.
[0067] The computer-controlled high-throughput sample mounting and
alignment system has been designed as a device optimized for
reliable removal of the samples from the liquid nitrogen sample
repository system and mounting on a sample positioner. The sample
positioner, as further described below, positions the sample after
it has been removed from the sample repository to a predetermined
position in space through which an x-ray beam will subsequently
pass, illuminating the sample and thereby generating
crystallographic diffraction patterns. The major subsystems of the
sample mounting and alignment system are: the sample positioner, a
coordinated set of translational and rotating stages for
positioning and orienting the sample gripper (known as the gripper
stage), a repository system, a gripper defroster, an optical sample
alignment system, and an x-ray camera. This system positions a
sample crystal at a precise point in space for x-ray imaging
analysis, or crystallography.
[0068] The sample repository system is principally a liquid
nitrogen filled storage Dewar removably placed atop a
position-addressable repository stage. It accommodates up to four
sample cassettes on a cassette deck corresponding to 64 samples per
load. The individual sample assemblies are held fixed in the
storage Dewar by a machinable magnetic material that attracts and
retains the ferromagnetic material in the sample assembly. The
storage Dewar is mounted on a two axis movable platform that
provides alignment of any one of the 64 samples on the cassette
deck 700 (FIG. 7A) beneath the sample gripper (described below). By
using a larger Dewar, more sample assemblies can be tested in one
loading. Loads of 300 or more sample assemblies may be used.
[0069] In the preferred embodiment, the repository stage has one
rotational degree of freedom, and one linear degree of freedom. By
using a rotary stage atop a linear stage, valuable real estate is
conserved, as the stage never has to move more than half of the
diameter of the (preferably axisymmetric) storage Dewar.
[0070] The storage Dewar itself has external position referencing
features that precisely and removably locate the storage Dewar atop
the rotary and linear stages. The storage Dewar also has an
internal referencing system that is aligned with the external
position reference features. A removable cassette deck is placed
through alignment pins in the internal referencing system. In this
fashion, by placing the storage Dewar on the repository stage,
individual sample assemblies mounted on the cassette deck can be
moved to a specific location required for sample pickup and
subsequent mounting. Samples can likewise be moved from one
position to another (empty) position either on the same or
different sample cassette.
[0071] The sample gripper contains a liquid nitrogen cooled split
collet mechanism for grasping the sample assembly with sufficient
force to overcome the magnetic attraction between the sample
assembly base and the sample cassette base with its magnetic
material, as well as to break free of any ice binding between them.
The sample gripper is designed to encapsulate the sample tube with
the sample crystal-containing sample loop within a liquid nitrogen
temperature environment during the brief period when the gripper
stage uses the sample gripper to transfer the sample assembly to
the sample positioner.
[0072] The sample gripper contains a low thermal capacity
(preferably very thin stainless steel) outer shroud, which provides
a sheath-flow of warm dry gas to prevent icing and frost formation
during exposure to ambient temperature, moisture-laden air. After
the sample assembly is mounted, the sample is maintained in a
cryostream of dry, near liquid nitrogen temperature gaseous
nitrogen during subsequent crystallographic measurements.
Sufficient cryostream flow is provided to maintain the sample at
liquid nitrogen temperatures despite beam heating induced by the
probing synchrotron-produced x-ray heat load and closely juxtaposed
ambient atmosphere.
[0073] Removal of the sample assemblies from the sample positioner
and replacement in the sample repository is accomplished using the
same sample gripper. After removal, the mounting post upon which
the sample assembly was just previously mounted could accumulate
frost, interfering with the magnetic retention of the next sample
assembly. If this frost becomes problematic, the mounting post
could be actively heated.
[0074] The sample gripper is periodically warmed to remove any ice
accumulation. The gripper defroster provides either heated dry
nitrogen gas or heated dry air to the outer shroud of the gripper
through a pattern of small holes. Due to the low thermal mass of
the outer shroud of the sample gripper (recall that it is
preferably very thin stainless steel), defrost typically occurs in
6 seconds or less. The upper part of the sample gripper is
furthermore heated, allowing the sample gripper to be left in the
liquid nitrogen Dewar indefinitely to keep it cold, but at the same
time not damaging the pneumatic actuator mounted on top operating
at near room temperature, which cannot be exposed to cryogenic
temperatures.
[0075] The gripper stage moves the sample gripper through the
mechanical motions used for removal and placement of the sample
assembly between the sample positioning stage and the sample
repository. The gripper stage comprises two orthogonal pneumatic
translational motions (XY) (which could also be motor-controlled)
upon which is mounted a third rotational (.theta.) motion
mechanism. A relatively low force pneumatic actuator, mounted on
the rotational motion mechanism, provides further independent
vertical movement. The low force pneumatic actuator prevents damage
from the sample gripper impacting misplaced sample assemblies or
frost.
[0076] The gripper stage motions are preferably based in part on
pneumatic actuators to achieve precise and rapid motions in the
liquid nitrogen cooled environment. The three-degree of freedom,
(XY.theta.) positioner of the gripper stage has been designed to
move the sample gripper with the required positioning precision
within the restricted allowable physical envelope of the
experimental setup. Electrically powered motors and actuators could
potentially replace some or all of these pneumatic actuators,
however, pneumatic actuators have proven to be simpler, cheaper,
and easier to control and maintain in this application.
[0077] The sample positioner is a three-degree of freedom stage,
mounted on a precision goniometer to provide single axis rotation.
The three-degree of freedom stage has been custom designed to
position a sample assembly mounted on a mounting post at a precise
position in the synchrotron x-ray beam, and, in coordination with
the goniometer, to rotate the sample about that point. The three
degree of freedom stage consists of two orthogonal motor-controlled
translational motions (XY) upon which is mounted a third Z axis
motion mechanism. By coordinating the motions of the XY stage and
the goniometer, a mounted sample can be rotated about a defined
point in space despite initial eccentric mounting. The overall
hysteresis of the three degree of freedom stage is about 1.5
microns with a stability of about 1 micron. The system has been
shown to be superior to existing commercial versions with respect
to position precision and stability.
[0078] The optical alignment system that provides feedback for
precise alignment of the sample is based upon a high-resolution
zooming camera that is optically aligned to a point in space that
will be subsequently illuminated by the synchrotron x-ray beam. A
software reference pixel location for the center of the x-ray beam
is initially established. Visual images of the mounted crystal
sample are then compared with this reference. Any necessary
positional corrections are calculated and executed by the sample
positioner to position the sample over the software reference
position.
[0079] The software and user interface designs have been structured
to accommodate alternative centering feedback data based, for
instance, on the measured x-ray intensities or data quality as
indicated by the x-ray data collection imaging device.
[0080] It should be noted that the system described herein could be
used for positioning cryogenically stored samples to a particular
point in space for many alternative types of measurements. These
measurements may be done by observing the sample at a single
prescribed point in space, or by observing the sample at a variety
of rotational angles as it is rotated by the goniometer. Depending
on the relative time durations of the sampling measurement, the
sample may be statically paused at each measurement, or may be
continually rotated. An example of a continual rotation measurement
could be that of short pulse-width laser illumination using short
pulses having microsecond, nanosecond, or picosecond pulse-widths.
With such short pulse-widths, even a moving sample appears as if it
were stationary.
[0081] Data collection may be used on any experimental device
outputting information that can be used as a measurement. Examples
include, but are not limited to: x-ray crystallography,
photonics-based tomography, photonics-based diffraction, surface
spectroscopy, fluorescence correlation spectroscopy, photon arrival
time interval distribution analysis, fluorescence resonance energy
transfer methods, mass spectrometry, and evanescent wave methods,
scanning probe microscopy, taccimetry, profilometry, and atomic
force microscopy.
Sample Assembly
[0082] Referring now to FIG. 1, a sample assembly 100 is comprised
of a sample 110 captured in a sample loop 120. The sample 110 is
preferably frozen at cryogenic temperatures, such as that of liquid
nitrogen at standard atmospheric pressure, -195.79.degree. C. or
77.36.degree. K. The sample loop 120 is in turn attached by
adhesive 130, preferably alpha cyanoacrylate, to sample tube 140.
The sample tube 140 is attached to sample base 160 by insertion
into hole 150. The sample base 160 has an upper land 180 for
retention, and a lower recess 170 for mounting.
[0083] Sample base 160 is ferromagnetic, preferably easily
free-machining ANSI 1118 steel with zinc electroplating after
machining for corrosion resistance. The sample tube 140 is
preferably stainless steel or other low thermal conductivity
material, preferably already containing an attached 50 to 1000
.mu.m nylon sample loop 120.
Sample Cassette
[0084] Referring now to FIGS. 2A, 2B and 2C, a sample cassette 200
holding one sample assembly 100 is shown. The sample cassette 200
has a cassette base 210 into which a keyed shaft 220 is affixed on
one end 225. A magnet 250, preferably a machinable magnet of
polymeric matrix material, is inserted into the cassette base 210,
preferably in a recess 255 in the cassette base 210. The machinable
magnet 250 has one opening 230 aligned with each corresponding
cassette base opening 265. Sample assemblies 100 are then able to
be loosely inserted into cassette base 210 through openings 265 and
be retained by the upper surface 260 of magnet 250, with cooling
liquid nitrogen able to flow through each magnet 250 opening 230
and cassette base 210 opening 265 to directly contact, and hence,
cool the sample assembly 100 through direct contact with lower
recess 170 (shown in FIG. 1).
[0085] In this embodiment of the sample cassette 200, 16 sample
assemblies can be accommodated in a regular pattern forming two
concentric circles. However, any other regularized or
nonregularized pattern (not shown in FIG. 2B) could be used as
well.
[0086] Keyed shaft 220 is present to align a cassette cover 300
(described below) so that it does not damage the samples 110 when
they are covered for transport or storage. Many other functionally
equivalent methods, using multiple unkeyed shafts in a pattern, or
external jigs, would also achieve the result that cassette cover
300 placement onto the sample cassette 200 would not damage any of
the samples 110. The attachment of keyed shaft 220 is preferably by
a threaded screw into a threaded recess in the keyed shaft,
however, many other mechanically equivalent attachment methods
would also work, including, but not limited to press fit, shrink
fit, adhesive attachment, welding, or threading.
Sample Cassette Cover
[0087] Referring now to FIGS. 3A, 3B and 3C, a sample cassette
cover 300 is a cylindrical shape with a keyed opening 310 passing
through a threaded section 330. A cylindrical neck 340 portion
protrudes above the main top surface 320, and is centered on the
sample cassette cover 300 center 305. An indexing feature 350
serves as clearance for an alignment feature to be discussed
below.
[0088] In the sample cassette cover 300 bottom surface 355,
pluralities of sample assembly recesses 360 provide protection to
samples assemblies 100 (shown in FIG. 1) placed in them. In this
embodiment, an outer vent ring 370 and an inner vent ring 380 vent
the sample assembly recesses 360; these details are not shown with
hidden lines in FIG. 3A to minimize confusion of hidden lines. The
outer vent ring 370 and an inner vent ring 380 are respectively
ported to the exterior of the sample cassette cover 300 with
pluralities of outer vent ports 375 and inner vent ports 385. This
venting arrangement allows for the flow of liquid nitrogen to each
of the sample assembly recesses 360, ensuring that sample
assemblies 100 (shown in FIG. 1) as well as the cassette cover 300
are amply cooled by liquid nitrogen. It also allows the venting of
nitrogen gas, which might otherwise build up inside the sample
assembly recesses 360.
Sample Cassette Assembly
[0089] Referring now to FIG. 4A, a sample cassette assembly 400 is
shown comprised of a sample cassette cover 300 assembled in place
over a sample assembly 100, and retained by sample cassette 200.
Now referring to both FIGS. 4A and 4B, sample assembly 100 is
magnetically retained on the upper surface 260 of magnet 250.
[0090] During installation, the sample cassette cover 300 first
encounters keyed shaft 220. The sample cassette cover 300 must
first be rotationally aligned relative to sample cassette 200 so
that the keyed shaft 220 may translate into the keyed opening 310.
Since the keyed shaft 220 is taller than the installed sample
assembly 100, the sample 110 cannot be damaged by the sample
cassette cover 300 during normal installation of the sample
cassette cover 300 as sample assembly recesses 360 in the sample
cassette cover 300 slides over each of the sample assemblies
100.
[0091] The sample cassette cover 300 has one sample assembly recess
360 for each matching cassette base 210 opening 265. The depth of
the sample assembly recesses 360 exceeds the retained height of the
sample assembly 100. Additionally, the sample assembly recesses 360
have smaller diameters than the width of the sample assembly 100,
so that the bottom surface 355 of the sample cassette cover 300
positively retains the sample base 160 by contacting upper land
180.
[0092] When assembled as shown in FIG. 4A, the sample assemblies
100 are positively sandwiched between the sample cassette cover 300
and the sample cassette 200. Now referring additionally to FIG. 3C,
the outer vent ring 370 and the inner vent ring 380 allow for
filling of the sample assembly recesses 360 with liquid nitrogen
through outer vent ports 375 and inner vent ports 385.
[0093] In one embodiment of the invention, the assembled cassette
cover 300 and sample cassette 200 are inverted so that sample
assembly recesses 360 form liquid nitrogen repositories, thus
keeping the biological crystal sample 110 immersed in liquid
nitrogen and maintaining the sample at a cryogenic temperature
until all of the liquid nitrogen has boiled away. In this
embodiment, several minutes of room temperature exposure can be
tolerated by the sample with minimal temperature rise when moving
samples from shipping container to cryogenic sample repository.
[0094] Additional mechanical components (not shown) clip and retain
the sample cassette cover 300 to the sample cassette 200, although
many other methods of positively retaining the parts together
exist, and are readily designed by those skilled in the mechanical
design arts.
Sample Cassette Carrier
[0095] Refer now to FIGS. 5A and 5B, where a cassette carrier 500
is depicted. A top hook 510 is press fit into a low thermal
conductivity sleeve 515, preferably comprised of fiberglass or
other low thermal conductivity low temperature plastic, which has
inserted into it a stainless steel rod 520. The stainless steel rod
520 is welded to a tab 525 formed by two narrow slots 530 on either
side. The tab 525 is part of a sheet 580 that encompasses, and is
attached to, about half the diameter of a plurality of shelves 550.
The method of attachment could be any that survives repeated
thermal shocks from room temperature to liquid nitrogen
temperature. In this embodiment, three screws 590 are used to
attach each shelf 550 to the sheet 580.
[0096] The distances between the shelves 550 form a set of shelf
openings, or landings 535. For purposes of illustration, the
top-most landing 535 is vacant, without a sample cassette assembly
400. The other six shelve openings in the diagram each show sample
cassette assemblies 400 present.
[0097] In FIG. 5B, each shelf 550 has an inner 551 and outer 552
radius concentric about a center point 555, which is the same
center point for a mounted sample cassette assembly 400. During
insertion of the sample cassette assembly 400, cylindrical neck 340
(shown in FIGS. 3A and 3B) of the sample cassette cover 300 slides
into obround slot 560, where retaining spring 570, secured by
fastener 575, retains the sample cassette cover 300. Retaining
spring 570 deflects partially into retaining spring recess 565.
When fully inserted, cassette cover 300 center 305 is roughly
concentric with center point 555.
Cryogenic Transport Container
[0098] Before shipping, the cryogenic transport container 600 of
FIG. 6 is initially precooled with liquid nitrogen for shipping per
the manufacturer's directions. Some of these precooling steps can
take as long as four hours to complete.
[0099] Referring now to FIG. 6, cryogenic transportation container
600 has a removable insulation plug 610, which inserts into a
corresponding cylindrical bore 630 in bulk insulation 620. When
cylindrical bore 630 is initially empty except for nitrogen gas and
liquid, the cassette carrier 500, having one or more sample
cassette assemblies 400, is first inserted. Subsequently, the
removable insulation plug 610 is installed. Further liquid
nitrogen, if needed, is added per the manufacturer's directions.
Depending on the manufacturer of cryogenic transportation container
600, ambient temperature exposure, and the detailed construction of
the container, cryogenic temperatures below -150.degree. C. can be
maintained during shipment for up to 200 hours.
Cassette Deck
[0100] Refer now to FIGS. 7A and 7B. The cassette deck 700 is
shown. The cassette deck 700 has a center reference hole 710, three
mounting holes 760, and a larger diameter keying hole 750 to
uniquely orient the cassette deck 700 with respect to hardware
incorporated into the sample repository (not shown). Orientation
pins 720 are preferably press fit into the cassette deck 700. The
orientation pins 720 provide a unique orientation of sample
cassettes 200 which are positioned between the two other outer
positioning pins 740. This pattern is replicated in four quadrants.
In one quadrant there is just an outline of the area 745 that is
normally occupied by a sample cassette 200.
[0101] By using this arrangement, in conjunction with the sample
cassette 200 design, all sample assemblies 100 are uniquely
positioned with respect to the cassette deck 700. The unique
positioning allows for unattended sample assembly 100 mounting and
demounting using a sample gripper described below.
Sample Gripper
[0102] The sample gripper 800 is shown in FIG. 8. An upper actuator
flange 865 is connected to a lower actuator flange 805. The actual
mechanism of the actuator is not shown, as these are readily
commercially available as either solenoidal electrical or pneumatic
force/displacement devices. The preferred actuator is pneumatic.
The lower actuator flange 805 has been modified so that in
conjunction with gripper flange 815, a port 810 is formed. The port
810 attaches to a plenum 812. The plenum 812 allows a continuous
gas connection with a series of cylindrical openings 820, which at
their apex, connect to small openings 825. Input gas can be
attached to port 810, fill plenum 812, pass through a plurality of
cylindrical opening 820, and emit at small openings 825 into an
outer shroud area 832. The outer shroud area 832 is formed by an
inner tube 830 and outer tube 835, which are both attached to
gripper flange 815. The outer tube 835 necks down to a removable
close fitting shroud tube 840. The inner tube 830 has a very low
thermal mass, low heat capacity material, and is preferably both
very thin walled, and made of stainless steel. At the lower end of
the inner tube 830, is attached a collet sleeve 845. The collet
sleeve 845 is preferably silver soldered (not shown) to the
stainless steel inner tube 830. The inner tube 830 must be
sufficiently thick so as to keep from bucking under axial
compressive forces generated by the collet sleeve 845.
[0103] The split collet 850 has an actuation movement relative to
the collet sleeve 845, closing the split collet 850 about a sample
assembly 100 located within its grasp. When split collet 850 is
retracted upwards, the collet sleeve 845 causes compressive closure
of the collet actuation surface 846, with consequent high force
retention of the sample assembly 100 located within the split
collet 850 in a collet recess 847. The split collet 850 is pulled
upward by collet adapter 854, which connects the split collet 850
to the collet tube 855. The collet tube 855 is in turn connected to
the actuator adapter 860. The actuator adapter 860 connects collet
tube 855 to the actuator tube 870. Thus a vertical motion of the
actuator tube 870 causes the same vertical motion of the actuator
adaptor 860, collet tube 855, collet adapter 854, and in turn the
split collet 850.
[0104] The temperature of the split collet 850 is measured by a
temperature sensing element 852 located at the bottom of a
temperature sensing hole 851. Wires (not shown to minimize drawing
clutter) ascend upward through the temperature sensing hole 851,
through a matching hole in collet adapter 854, and exit the sample
gripper 800 through the center bore 875 of actuator tube 870.
[0105] To cool the sample gripper 800 down to temperatures
appropriate for sample assembly 100 pickup (e.g. liquid nitrogen
temperature), the collet sleeve 845 end of the sample gripper 800
is immersed in liquid nitrogen. At this time, there is no sample
assembly 100 present. A small gage vacuum of 3-4 inches of mercury
is drawn on port 810, which is communicated through the small
openings 825 to the outer shroud area 832. Vent port 833 in inner
tube 830 allows the vacuum to pull liquid nitrogen up and around
split collet 850. Since the collet is split, liquid nitrogen fills
the interior 853 of the split collet 850. The temperature-sensing
element 852 is used to register when the split collet 850
temperature has cooled sufficiently for sample assembly 100
pickup.
[0106] When the sample gripper 800 is moving the sample assembly
100, room temperature dry nitrogen gas is fed through port 810 into
the outer shroud area 832 to preclude frost buildup on inner tube
830 or split collet 850. The frost buildup is prevented by the
simple expedient of keeping moisture away from any of the cold
surfaces of the inner tube 830 or split collet 850, by the flow of
the dry nitrogen gas, preferably in laminar flow.
[0107] The sample gripper 800 is drawn showing most details, with
the center section abbreviated by a cut 890.
Integrated Crystal Mounting and Alignment System for
High-throughput Biological Crystallography
[0108] Referring now to FIG. 9A, we see the integrated crystal
mounting and alignment system for high-throughput biological
crystallography 900 as viewed down an axis parallel to the incoming
synchrotron x-ray beam 901. A frame 902 connects the various
subsystems, and will not be fully described other than to say that
it must be sufficiently stable and stiff to keep most components
accurately positioned to within about 1 .mu.m.
[0109] The subsystems include a repository stage, a gripper stage,
a sample positioner, a cryostream unit, a video alignment
subsystem, and a collimation and beam blocking subsystem. These
subsystems are more fully described sequentially below.
[0110] The repository stage is comprised of the Y.sub.1 linear
stage 904, which is mounted on the frame 902. Atop the Y.sub.1
linear stage 904 is attached rotary stage 906, which rotates about
a vertical axis of revolution. Storage Dewar 908 removably attaches
to the rotary stage 906 at a repeatable position and orientation
using standard mechanical precision engineering fixturing
techniques that are well known in these arts. The storage Dewar 908
is nominally filled with enough liquid nitrogen 910 to amply cover
any sample assemblies 100 that may be present in any sample
cassettes 200. The cassette deck 700 is mounted on position
referencing components not described here, which allows sample
cassettes 200 to be addressably positioned relative to the frame
902 with high accuracy.
[0111] The gripper stage moves the sample gripper 800 relative to
the frame 902. It comprises a horizontal stage 926, an UpDown stage
924, and a rotary stage 920. A long travel Y.sub.2 linear stage,
known as the horizontal stage 926, moves horizontally (along the
plane of the paper) a vertical stage mounted transversely thereon,
known as UpDown 924. The UpDown stage 924 comprises a platform 922
that serves as a mounting base for a 90.degree. rotary stage 920,
known as Rotary, preferably a pneumatic 90.degree. rotary stage.
The 90.degree. rotary stage 920 top mounts a small travel, lighter
actuation force pneumatic stage called SmallMove 918, which serves
as a mount for the sample gripper 800. Rotary stage 920 rotate the
sample gripper 800 between a downward position (as shown in FIG.
9C) and a horizontal position (as shown in FIG. 9D).
[0112] The sample positioner is comprised of a high precision
rotary stage known as a goniometer 928, which is mounted on the
frame 902. The goniometer 928 rotates an angle .theta. (theta)
along an axis typically parallel to the horizontal plane. Upon the
goniometer 928 is mounted a. A Z' stage 932 with a magnetic
mounting post 934 mounts onto the X'Y' stage 930. At the particular
goniometer 928 angle .theta. depicted in FIGS. 9A-9G, the X'Y'
stage 930 moves in and out of the plane of the paper (the X' axis),
and up and down in the plane of the paper (the Y' axis). These
motions will rotate with continued rotations of the goniometer 928
in a typical kinematic rotating frame of reference. The mounting
post 934 is mounted upon, and moved by, compound X'Y' stage 930.
The mounting post 934 is spring preloaded (to prevent hard sample
assembly 100 mountings), and rides on two sets of three bearings,
each set of which forms an equilateral triangle.
[0113] In an alternate embodiment of the system, the sample
positioner is further comprised of a tilt plate 929 (as depicted in
FIG. 10A) disposed between the high precision rotary stage known as
the goniometer 928, and the compound X'Y' stage 930, to which the
Z' stage 932 is attached as before. The effect of the tilt plate
929 is to rotate the Z' stage 932 eccentrically with respect to the
axis of rotation of the goniometer 928 so that the positioner
rotates the sample 110 about an axis non-orthogonal with the
compound X'Y' stage 930 and the Z' stage 932. The tilt plate 929
preferably forms a tilt angle of at least 15.degree., more
preferably of at least 10.degree., yet more preferably of at least
5.degree., still more preferably of at least 2.degree., and most
preferably of at least 1. The alignment and centering operations
described below may be used either directly by ignoring the effect
of the tilt plate 929, or by including the angle of the tilt plate
929 in the alignment and centering algorithms. Regardless of the
eccentricity induced by the tilt plate 929, the properly centered
sample 110 will maintain location within the x-ray synchrotron beam
901 when the beam is operational, and the same spatial position
when the x-ray synchrotron beam 901 is non-operational, as further
described below. Rotations of the sample 110 will normally require
operation of the compound X'Y' stage 930 and the Z' stage 932 for
correct positioning.
[0114] In yet another embodiment (shown in FIG. 10B) another tilt
plate 931 may be disposed between the compound X'Y' stage 930 and
the Z' stage 932 to effect the eccentricity described above. In
this further embodiment, the other tilt plate 931 would preferably
form a tilt angle of at least 15.degree., more preferably of at
least 10.degree., yet more preferably of at least 5.degree., still
more preferably of at least 2.degree., and most preferably of at
least 1.degree..
[0115] In operation, a sample assembly 100 (already removed here,
and thus not shown) is retained by the mounting post 934 by
magnetic attraction. The mounting post 934 could readily be heated
to prevent frost formation, but heating has not yet proven
necessary. The coordinated motions of the rotation addressable
goniometer 928, the compound X'Y' stage 930 and the Z' stage 932
allow a sample to be rotated in space about a predetermined point,
preferably the incoming synchrotron x-ray beam 901.
[0116] A commercially available cryostream unit 936 emits a stream
of near liquid nitrogen temperature nitrogen gas to cool the sample
110 when the sample assembly 100 is mounted on the mounting post
934. The cryostream unit 936 is actuated along axis 938 so as to:
1) prevent interference with the sample gripper 800 when mounting
or unmounting sample assemblies 100 (not shown), 2) not optically
occlude the camera 948 optical aperture, and 3) not interfere with
the projection of the incoming synchrotron x-ray beam 901,
regardless of whether or not the x-ray beam 901 is operational.
[0117] The video alignment subsystem is comprised of a commercially
available backlighter 956 on a vertically extendible backlighter
stage 958. During alignment, the backlighter stage 958 raises the
backlighter 956 so that the sample 100 (not shown, but mounted on
mounting post 934) is back-lit when viewed by the high resolution
macroscopic zooming video camera 948. During x-ray irradiation, the
backlighter 956 is retracted so that it is out of the direct x-ray
beam 901.
[0118] Collimation and beam blocking is typically required to
respectively form a parallel incoming x-ray beam of a controlled
diameter, or stop the beam altogether. For collimation to work
properly, a small aperture must be aligned with the incoming x-ray
beam. Collimation and beam blocking of the x-ray beam is effected
by using the collimator vertical actuator 940 to raise a
piezoelectric actuator 942, to which an x piezoelectric actuator
944 is attached, which moves a selection of collimators having
various diameters and beam blocks 946 into the x-ray beam 901. Note
that the collimators and beam blocks 946 with their associated
actuators, are in a non-interfering plane from the backlighter 956
so that each may operate independently without collision.
[0119] To collimate or locally block the synchrotron x-ray beam
901, the various diameter collimators and beam block 946 is moved
up into the x-ray beam 901 by the collimator vertical actuator 940,
and is precisely positioned for optimal collimation by small,
precise movements effected by piezoelectric actuators 942 and
944.
Application of the Invention to Mount a Sample Assembly
[0120] Refer now to FIGS. 9A-9F, which is a sequence of partial
front views of the integrated crystal mounting and alignment system
for high-throughput biological crystallography 900 with most of the
major subsystems illustrated. The sequence of FIGS. 9A-9F show some
of the major steps involved in conveying a sample assembly 100 to
the sample positioner mounting post 934 for alignment using camera
948 and subsequent data collection from crystallographic
diffraction of the incoming synchrotron x-ray beam 901 by the
sample 110 (not shown).
[0121] Initially, the sample gripper 800 must be cooled
sufficiently to safely grasp a sample assembly 100. The sample
gripper 800 is initially partially immersed in the liquid nitrogen
910 so that the temperature-sensing element 852 (shown in FIG. 8)
is cooled to a temperature of at least -150.degree. C. prior to
continuing with the sample assembly pickup. This initial sample
gripper 800 immersion is located away from any resident sample
assemblies 100 present in the storage Dewar 908. For this purpose
the sample gripper 800 is immersed in the storage Dewar 908 until a
set temperature is achieved. This initial cooling procedure
typically requires over a minute. However, in normal operation, the
cooling down procedure is only required once in a set of samples
since the sample gripper 800 remains cold with repeated immersions
in the liquid nitrogen 910. When a sample assembly 100 pickup
occurs, further additional cooling will take place as the sample
gripper 800 is immersed in the storage Dewar 908.
[0122] FIGS. 9A-G correspond to the integrated crystal mounting and
alignment system 900 moving through a sequence of configurations as
described more fully below. It is appreciated that there are many
alternative sequences and minor variations that may be used to
effect the same operations.
[0123] In FIG. 9A, the system is in the process of using the sample
gripper 800 to grasp a sample assembly 100 in the storage Dewar
908. From there, it will move the sample assembly 100 to the sample
positioner mounting post 934 for alignment and x-ray probing. At
this step in the protocol, a sample assembly 100 has been grasped
by sample gripper 800. Prior to grasping the sample assembly 100,
the following setup steps have occurred: (1) the gripper 800 is
released; (2) the heater 950, collimator beam blocks 946, the
cryostream unit 936 are retracted so as to not interfere with other
movements; (3) Rotary 920 is rotated down so that the sample
gripper 800 assumes a vertical orientation; (4) UpDown 924 has been
moved down; (5) SmallMove 918 has been downwardly extended; and (6)
the gripper stage has moved the sample gripper 800 to a location in
the storage Dewar 908, and immersed the sample gripper 800 in the
liquid nitrogen 910 until the sample gripper 800 has reached a
temperature of -130.degree. C. as measured by the temperature
sensing element 852 (indicated but not shown in FIG. 8). Gripping
is accomplished by moving the sample gripper 800 over a selected
sample assembly 100 located in a predefined location pattern in the
storage Dewar 908. The sample gripper 800 is actuated, causing the
split collet 850 to exert a pressure on the sample assembly 100.
The friction generated by this pressure is sufficient to overcome
the frost buildup and/or magnetic attraction of the sample assembly
100 to the sample cassettes 200 in the storage Dewar 908.
[0124] Next, in FIG. 9B, the system has retracted the SmallMove 918
pneumatic stage, causing the sample gripper 800 to be moved
vertically upwards. Depending on the liquid nitrogen 910 fill level
in the storage Dewar 908, the sample assembly 100 may have cleared
the liquid nitrogen 910 surface, as depicted here. Not shown in the
drawings is an automated liquid nitrogen fill apparatus, to keep
the liquid nitrogen 910 fill level at a specified level. The
storage Dewar 908 is typically nearly full, so that the sample
gripper 800 is partially immersed even when SmallMove 918 is
retracted to its highest vertical position.
[0125] Next, in FIG. 9C, the system has actuated the transverse
vertical stage, known as UpDown 924, causing the platform 922 to
move further vertically upwards, carrying the sample gripper 800
vertically upwards, so that the grasped sample assembly 100
vertically clears the top of the storage Dewar 908.
[0126] Next, in FIG. 9D, the system has rotated the pneumatic
90.degree. rotary stage 920, known as Rotary, causing the sample
gripper 800 and sample assembly 100 to rotate 90.degree. clockwise
and point toward the sample positioner mounting post 934. Sample
assembly 100 is essentially collinear with mounting post 934.
[0127] Next, in FIG. 9E, the sample gripper 800 has moved by the
long horizontal travel linear stage, known as the Y.sub.2 stage
926, causing the sample gripper 800 to move to a predetermined
distance toward the sample positioner mounting post 934, in a
vector parallel to the Z' stage 932 motion (horizontally as
indicated in FIG. 9E), with sample assembly 100 approaching the
mounting post 934 in advance of sample gripper 800.
[0128] Next, in FIG. 9F, the SmallMove 918 pneumatic stage has been
extended in a short horizontal translation to mount the sample
assembly 100 gently in contact with the magnetically attractive
mounting post 934 of the sample positioner. After contact with the
mounting post 934 is accomplished, the gripper assembly 800
releases its grip on the sample assembly 100. Now the cryostream
unit 936 is actuated along line 938 to approach the sample 110
mounted on the sample assembly 100 mounted on the mounting post
934. The cryostream unit 936 is then activated to cool the sample
assembly 100 sample 110 (still cryogenically shielded in the
gripper assembly 800) which is now roughly located at a spatial
position where the x-ray beam 901 will subsequently irradiate.
[0129] Next, in FIG. 9G, the sample gripper 800 has been moved a
predetermined distance away from the sample positioner mounting
post 934 by the long travel Y.sub.2 linear stage, known as the
horizontal stage 926. Since the sample gripper 800 has already been
released, the sample assembly 100 remains on the sample positioner
mounting post 934 due to magnetic attraction. Since the cryostream
unit 936 has already been activated, the sample 110 is released
from the sample gripper 800 cryogenic interior directly into the
cold stream of the cryostream unit 936. In this manner, the sample
110 is never exposed to ambient room temperature.
Initial Sample Reference Position Setup
[0130] In this system, a zooming microscopic camera 948 views
provides information to correctly position the mounted sample 110
(shown earlier in FIG. 1). In order to establish this spatial
position, a three-dimensional position in space must be aligned
with the incoming synchrotron x-ray beam 901 while the beam is
active. With the x-ray beam 901 active, a pin, or other small
axisymmetric alignment shape is moved into the beam until the beam
is partially occluded. An x-ray imaging camera, operationally
similar to the high resolution macroscopic zooming video camera
948, except that x-rays are detected, and zooming is not likely
necessary, is used to image the axisymmetric alignment shape.
Either the synchrotron beam current must be greatly reduced, or
more preferably the x-ray beam intensity is greatly attenuated by
an attenuator to prevent burnout of the camera for this
operation.
[0131] With the x-ray beam 901 producing an image on the x-ray
imaging camera, the axisymmetric alignment shape (typically a small
bead, or pin with a point) is moved by coordinated movements of the
X' and Y' compound stage 930, and Z' stage 932 axes. Eventually,
the movements are manually (or possibly computer controlled)
coordinated until the alignment shape enters the field of view of
the x-ray imaging camera. By coordinated movements of the X' Y'
compound stage 930, and Z' stage 932, and rotation of the
goniometer 928, a three-dimensional reference location for the
center of the x-ray beam relative to the X' Y' compound stage 930,
and Z' stage 932 is developed. Once the alignment shape is aligned
to the x-ray beam, the beam may be turned off, or blocked
completely, as it is no longer necessary for the initial alignment,
as the x-ray beam center relative to the X' Y' compound stage 930,
and Z' stage 932 is already known.
[0132] The backlighter stage 958 now raises the backlighter 956 so
that the alignment shape is back-lit when viewed by the high
resolution macroscopic zooming video camera 948. Since the x-ray
beam may now be turned off, it is safe for personnel to enter the
potentially irradiated hutch area to manually (or by remote control
of appropriate tilt or pointing actuators) align the camera 948 to
view the alignment shape. Once the camera 948 is correctly
positioned to view the alignment shape in roughly the center of
field of view, no further camera 948 alignment should be necessary.
The camera 948 is then used to view the alignment shape. The
alignment shape is viewed, and the pixel location corresponding to
the portion of the alignment shape previously positioned in the
center of the x-ray beam is recorded. This is the pixel location of
the beam center at a particular zoom magnification. The zoom
magnification is then increased to a higher magnification so as to
more completely fill the field of view of the camera 948, and the
pixel location again corresponding to the portion of the alignment
shape previously positioned in the center of the x-ray beam is
recorded.
[0133] Note that the zooming camera 948, can typically only
determine position in two dimensions as imaged pixel locations.
Typical imaging devices can only focus within a particular optical
depth of field, which, depending on the depth of field of the
optical image, can provide additional information regarding a
distance from the optical objective by either being in or out of
focus. In this instance, the zooming camera 948 is preferably
parfocally focused on the alignment shape; so that it remains in
focus at all zoom magnifications.
[0134] Note that, at this time, there are two reference positions
being used: the software pixel location of the sample as viewed by
the zooming camera 948, which is a two dimensional pixel reference
position related to the field of view of the zooming camera 948;
and the spatial center of the alignment shape relative to the
positioner, a three dimensional reference. These software pixel
locations, and the spatial position of the alignment shape relative
to the positioner which has previously be collocated through the
center of the x-ray beam, are subsequently used to rapidly align
samples for x-ray crystallography.
Sample Position Setup
[0135] Once the sample assembly 100 has been positioned on the
positioner as depicted in FIG. 9G, the sample 110 must be aligned
to be concentric with the x-ray beam 901 when it is activated. The
previously obtained software pixel locations (two dimensional
information) and the spatial center of the alignment shape relative
to the positioner (three dimensional information) are used to
correctly center the sample crystal 110 for x-ray crystallography.
These coordinates are cooperatively used to position the sample
crystal 110 relative to the positioner so that the sample crystal
110 may be rotated about the point where the x-ray beam 901 passes
when it is activated.
[0136] In this system, a zooming microscopic camera 948 initially
views the sample 100 (shown earlier in FIG. 1) at minimum
magnification, and produces video images of the mounted sample
crystal 110. The images are read by a video frame-grabber to
provide a digital image of the sample. By either manual or computer
algorithmic operation, the frame pixel coordinates of the center of
the sample may be determined. The sample positioner (comprised of
X' Y' compound stage 930, Z' stage 932, and the goniometer 928) is
then actuated to translate the sample 110 to the software reference
pixel location arrived at during initial zooming camera 948
alignment in a plane roughly (preferably within 45.degree., more
preferably within 30.degree., yet more preferably within
15.degree., and most preferably within 5.degree.) parallel to the
image plane of the camera 948. The sample positioner then rotates
the sample 110 through angular movement of the goniometer 928, and
the process is repeated. At each rotation, the translational
increments of the X' and Y' compound stage 930, and Z' stage 932
axes are recorded. Subsequently, these coordinates are used to
arrive at the true three-dimensional spatial center of the sample
110 crystal relative to the positioner. The entire process is then
optionally repeated at higher zoom magnification levels as
necessary. This sample 110 spatial center may be determined
relative to the positioner in as few as two rotations due to the
short depth of field of the camera 948 at maximum zoom.
[0137] The distance from the alignment shape center to the sample
110 center forms an offset vector. The offset vector is used to
coordinate the movement of the sample 110 by relative movements of
the X' Y' compound stage 930, and Z' stage 932 at each rotation. In
this manner, the sample 110 may be rotated in space through a point
collocated with the center of the x-ray beam 901 when it is
operated. The x-ray beam 901 is not allowed to strike the sample
110 during alignment, so as to minimize any synchrotron-produced
x-ray 901 heating or x-ray induced chemical degradation.
Sample X-Ray Crystallography
[0138] In the normal operation of the system, the sample positioner
is moved so that the sample 110 is positioned to a location where
synchrotron generated x-rays 901 will be emitted after sample 110
alignment. After the sample 110 is aligned as described above, the
synchrotron x-ray beam 901 is unblocked, allowing x-rays to
irradiate the sample 110, which can then be rotated to any
arbitrary angular position while remaining centered within the
x-rays beam 901. After x-ray crystallography is complete, the
synchrotron x-ray 901 source is again blocked or shuttered so as to
interrupt delivery of the x-ray beam, effectively turning off the
x-ray beam. This blocking and unblocking of the x-ray source is
important since the x-rays can induce damage to the crystalline
sample, thereby degrading the data collected.
Sample Dismounting
[0139] Following the FIGS. 9G-9A in reverse is essentially the
sequence of motions used by the unmounting protocol, where the
sample assembly 100 initially mounted on the sample positioner
mounting post 934 is finally replaced in the storage Dewar 908.
[0140] For the remaining sample assemblies 100, the sequence of
steps previously described is performed in reverse, from 9G (the
present data collection state), to 9F, 9E, 9D, 9C, 9B, and 9A where
the sample assembly 100 is replace in the Dewar 908. The sample
gripper 800 is retracted sufficiently to clear the sample
assemblies 100, but still partially immersed in the liquid nitrogen
910. The Y.sub.1 linear stage 904 and rotary stage 906 are actuated
to position the next sample assembly 100 beneath the sample gripper
800. At this point, the process repeats for sampling of the
remaining sample assemblies 100.
[0141] After a period of use, the sample gripper 800 may become
frost covered. A warm up or defrost protocol is used to remove any
accumulated frost from the sample gripper 800. Although the spatial
configuration for defrosting is not directly shown in any Figure,
it is readily visualized. During the defrost cycle, a heater 950 is
extended by an In/Out stage 952, and warm dry nitrogen gas is
emitted from the heater 950 onto the sample gripper 800, which has
previously been moved into position for defrosting.
CONCLUSION
[0142] All publications, patents, and patent applications mentioned
in this application are herein incorporated by reference to the
same extent as if each individual publication or patent application
were each specifically and individually indicated to be
incorporated by reference.
[0143] The description given here, and best modes of operation of
the invention, are not intended to limit the scope of the
invention. Many modifications, alternative constructions, and
equivalents may be employed without departing from the scope and
spirit of the invention. In particular, the sequence of motions
used in mounting and demounting sample assemblies 100 may be
re-sequenced in a myriad of permutations without deviating from the
general goal to be achieved so long as components and subsystems do
not destructively interfere with each other.
* * * * *