U.S. patent application number 09/372134 was filed with the patent office on 2002-05-09 for automated sample handling for x-ray crystallography.
Invention is credited to GREER, JONATHAN, JONES, RONALD B., MUCHMORE, STEVEN W., NIENABER, VICKI L., OLSON, JEFFREY A., PAN, JEFFREY Y..
Application Number | 20020054663 09/372134 |
Document ID | / |
Family ID | 23466846 |
Filed Date | 2002-05-09 |
United States Patent
Application |
20020054663 |
Kind Code |
A1 |
OLSON, JEFFREY A. ; et
al. |
May 9, 2002 |
AUTOMATED SAMPLE HANDLING FOR X-RAY CRYSTALLOGRAPHY
Abstract
Method and apparatus for mounting a sample comprising a crystal
for X-ray crystallographic analysis, a method for aligning a sample
comprising a crystal for X-ray crystallographic analysis, which
sample is mounted on a positioning device, and a method for
determining the structure of a sample containing a crystal by means
of X-ray crystallography.
Inventors: |
OLSON, JEFFREY A.;
(LIBERTYVILLE, IL) ; JONES, RONALD B.; (MUNDELEIN,
IL) ; NIENABER, VICKI L.; (GURNEE, IL) ;
MUCHMORE, STEVEN W.; (LIBERTYVILLE, IL) ; PAN,
JEFFREY Y.; (LAKE FOREST, IL) ; GREER, JONATHAN;
(CHICAGO, IL) |
Correspondence
Address: |
ABBOTT LABORATORIES
DEPT. 377 - AP6D-2
100 ABBOTT PARK ROAD
ABBOTT PARK
IL
60064-6050
US
|
Family ID: |
23466846 |
Appl. No.: |
09/372134 |
Filed: |
August 11, 1999 |
Current U.S.
Class: |
378/79 ;
378/73 |
Current CPC
Class: |
Y10T 436/25 20150115;
G01N 23/20 20130101 |
Class at
Publication: |
378/79 ;
378/73 |
International
Class: |
G01N 023/20 |
Claims
What is claimed is:
1. A method for mounting a sample comprising a crystal, said method
comprising the steps of: (a) providing a crystal holder containing
at least one crystal; (b) providing a tool capable of retrieving
said crystal holder, said tool movable by means of a robot; (c)
providing a positioning device for mounting said crystal holder so
that said crystal is in the path of a beam of X-rays; and (d)
activating said robot so that said tool retrieves said crystal
holder, transfers said retrieved crystal holder to said positioning
device, and mounts said transferred crystal holder on said
positioning device.
2. The method of claim 1, wherein said crystal holder is mounted to
said positioning device by means of screw threads.
3. The method of claim 1, wherein said crystal in said retrieved
crystal holder is shielded from air.
4. The method of claim 1, wherein said crystal in said retrieved
crystal holder is maintained at a temperature not in excess of
about 160.degree. K.
5. A method for aligning a sample comprising a crystal, said sample
mounted on a positioning device, said method comprising the steps
of: (a) providing a sample, said sample mounted on a positioning
device; (b) providing an apparatus capable of viewing said mounted
sample, whereby said apparatus is capable of imaging said mounted
sample and determining coordinates of said sample relative to a
reference position; (c) providing a source of power for adjusting
said positioning device linearly along three orthogonal axes and
rotationally about one of said three axes; and (d) activating said
source of power to cause said positioning device to be adjusted
such that said sample is positioned into the path of a beam of
X-rays, said adjustment of said positioning device being at a
plurality of angles, such that said sample is positioned within
said beam of X-rays at any angle of rotational adjustment.
6. The method of claim 5, wherein said viewing apparatus is a CCD
camera.
7. The method of claim 5, wherein said source of power comprises at
least one motor.
8. The method of claim 5, wherein said plurality of angles ranges
from 0.degree. to 90.degree..
9. The method of claim 5, wherein said positioning of said sample
involves a least squares fit of offset data collected along said
three orthogonal axes at said plurality of angles to a
equation.
10. The method of claim 9, wherein said equation for two of said
three orthogonal axes is V.sub.i=.DELTA.X cos .phi..sub.i+.DELTA.Y
sin .phi..sub.i where V.sub.i represents the vertical offset of the
centroid of the image of said sample at an angle .phi..sub.i, and
.DELTA.X represents the unknown offset of the centroid of the image
of the sample from the X-axis and .DELTA.Y the represents the
unknown offset of the centroid of the image of the sample from the
Y-axis.
11. The method of claim 10, wherein said equation for said third of
said three orthogonal axes employs a simple average of offset data
at said plurality of angles.
12. A method for conducting X-ray scatter analysis on a sample
selected from a plurality of samples, said sample containing a
crystal, said method comprising the steps of: (a) providing a
crystal holder containing at least a crystal, said crystal holder
contained in a storage cell; (b) providing a tool capable of
retrieving said crystal holder, said tool movable by means of a
robot; (c) providing a positioning device for mounting said crystal
holder so that said crystal is in the path of a beam of X-rays; (d)
activating said robot so that said tool retrieves said crystal
holder from said storage cell, transfers said retrieved crystal
holder to said positioning device, and mounts said transferred
crystal holder on said positioning device; (e) providing an
apparatus capable of viewing said mounted sample, whereby said
apparatus is capable of imaging said mounted sample and determining
coordinates of said sample relative to a reference position; (f)
providing a source of power for adjusting said positioning device
linearly along three orthogonal axes and rotationally about one of
said three axes; (g) activating said source of power to cause said
positioning device to be adjusted such that said sample is
positioned into the path of a beam of X25 rays, said adjustment of
said positioning device being at a plurality of angles, such that
said sample is positioned within said beam of X-rays at any angle
of rotational adjustment; (h) providing a beam of X-rays, said beam
aimed at said sample; (i) recording scattering of X-rays from said
sample; and (j) activating said robot so that said tool retrieves
said crystal holder from said positioning device and transfers said
crystal holder retrieved from said positioning device to said
storage cell.
13. The method of claim 12, wherein said crystal holder is mounted
to said positioning device by means of screw threads.
14. The method of claim 12, wherein said crystal in said retrieved
crystal holder is shielded from air.
15. The method of claim 12, wherein said crystal in said retrieved
crystal holder is maintained at a temperature not in excess of
about 160.degree. K.
16. The method of claim 12, wherein a computer is employed to
automate said method.
17. The method of claim 12, wherein a computer is employed to
record scattering of X-rays from said sample.
18. The method of claim 12, wherein said viewing apparatus is a CCD
camera.
19. The method of claim 12, wherein said source of power comprises
at least one motor.
20. The method of claim 12, wherein said plurality of angles ranges
from 0.degree. to 90.degree..
21. The method of claim 12, wherein said positioning of said sample
involves a least squares of offset data collected along said three
orthogonal axes at said plurality of angles fit to an equation.
22. The method of claim 21, wherein said equation is
V.sub.i=.DELTA.X cos.phi..sub.i+.DELTA.Y sin.phi..sub.i where
V.sub.i represents the vertical offset of the centroid of the image
of said sample at an angle .phi..sub.i, and .DELTA.X represents the
unknown offset of the centroid of the image of the sample on the
X-axis and .DELTA.Y the represents the unknown offset of the
centroid of the image of the sample on the Y-axis.
23. The method of claim 22, wherein said equation for said third of
said three orthogonal axes employs a simple average of offset data
at said plurality of angles.
24. A device for holding a crystal comprising: (a) a base; (b) an
attachment element projecting from said base; (c) a stem projecting
from said attachment element, said stem supporting a loop for
holding said crystal; and (e) at least one aperture in said
attachment element for allowing venting of said device, said device
capable of being attached to both a storage cell and a positioning
device.
25. The device of claim 24, wherein said base is cylindrical in
shape.
26. The device of claim 25, wherein said base comprises a notch for
locking said device to a sample rack.
27. The device of claim 24, wherein said attachment element is
cylindrical in shape.
28. The device of claim 24, wherein said pin is cylindrical in
shape.
29. The device of claim 24, wherein said device is attractable to a
magnet.
30. The device of claim 24, wherein said attachment element is
threaded.
31. An apparatus for retrieving a crystal holder from a storage
cell comprising: (a) a rotatable element capable of retrieving the
crystal holder from the storage cell; (b) a means for rotating a
rotatable element in a given direction of rotation when said
rotating means is in a locked mode; (c) a means for providing a
controlled amount of torque when said rotating means is slipping
relative to said rotatable element; and (d) a means for activating
said rotating means and said torque controlling means.
32. An apparatus for retrieving a crystal holder from a storage
cell, comprising: (a) a clutch having a cylindrical housing, said
housing comprising a bore surrounded by a wall; (b) a cylindrical
plunger capable of moving axially within said bore of said housing;
(c) said plunger having at least one elongated grove on the
exterior periphery thereof, said groove capable of receiving a
locking pin; (d) said housing having at least one aperture
extending through said wall thereof; (e) at least one spring pin
retained in said aperture, said pin capable of engaging said
elongated groove when said plunger is disposed in a first position
in said housing, said spring pin capable of disengaging said
elongated groove when said plunger is disposed in a second position
in said housing; (f) a means in said housing for resiliently
biasing said plunger toward said first position in said housing;
(g) an annular ring in contact with said interior wall of said
housing, said ring providing friction between an output flange and
a friction plate; and (h) a shaft attached to said plunger, said
shaft capable of transmitting torque to said friction plate, said
shaft further capable of moving axially with respect to the
friction plate.
33. The apparatus of claim 32, further including an input
flange.
34. The apparatus of claim 33, further including means for
providing axial compliance of said output flange relative to said
input flange.
35. A device for holding a plurality of samples, said device
comprising a plurality of storage cells, each of said storage cells
having a base and an opening, the area of said opening greater than
the area of said base, at least one side wall circumscribing said
base and said opening, said base being of sufficient area to allow
placement of a sample holder, said opening being of sufficient area
to allow ingress of a tool for retrieving said sample holder, said
base having attached thereto a means for locking said sample holder
to said device.
36. The device of clam 35, wherein said means for locking said
sample holder to said device is a locking pin.
37. The device of claim 35, wherein said base of said device is
ferromagnetic.
38. A device suitable for moving a crystal holder from a storage
cell to a positioning device, said moving device comprising an
elongated element having a first end and a second end, said first
end capable of being linked to a robot, said second end capable of
being coupled to said crystal holder, said device capable of
maintaining a crystal in said crystal holder at a temperature no
higher than about 160.degree. K when said second end is couple to
said crystal holder.
39. The device of claim 38, further including a vent to allow air
to escape from said device.
40. A device suitable for moving a crystal holder from a storage
cell to a positioning device, said moving device comprising an
elongated element having a first end and a second end, said first
end capable of being linked to a robot, said second end capable of
being coupled to said crystal holder, said device capable of
shielding said crystal holder from ambient air when said second end
is couple to said crystal holder.
41. The device of claim 40, further including a vent to allow air
to escape from said device.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] This invention relates to X-ray crystallography, and, in
particular, to methods and apparatus for mounting and aligning of
samples for X-ray crystallographic analysis.
[0003] 2. Discussion of the Art
[0004] X-ray crystallography is an established, well-studied
technique for providing a three-dimensional representation of the
appearance of a molecule in a crystal. Scientists have employed
X-ray crystallography to determine the crystal structures of many
molecules.
[0005] In order to perform an X-ray crystallographic analysis, a
sample of the crystal must be mounted onto a positioning device,
then carefully aligned so that the entire crystal is within the
diameter of the X-ray beam, and X-ray diffraction data collected at
a number of rotational angles. Because the typical sizes of
crystals and the diameter of the X-ray beam are in the range of 100
to 400 micrometers, the alignment requires a high degree of
precision. In addition, to ensure the integrity of crystals, the
crystals must be stored under liquid nitrogen and maintained at
temperatures near that of liquid nitrogen during the entire
mounting, aligning, and data collecting processes. Currently,
mounting and aligning of samples is performed manually.
[0006] A typical X-ray crystallography apparatus comprises an X-ray
generator, a detector, and a rotating spindle onto which a finely
adjustable head of the positioning device is mounted. Raw
diffraction data collected by the detector are input into a
computer for processing. The head of the positioning device allows
minute adjustments in two axes that are perpendicular to one
another and to the axis of the spindle. Some heads of positioning
devices also allow for angular adjustments in one or more axes. A
third axis of adjustment is provided by translation of the rotating
spindle in a direction that is orthogonal to the two axes of the
head of the positioning device. The sample mount position of the
head of the positioning device is positioned so that when mounted,
the sample is near the centerline of the X-ray beam. A CCD camera
is mounted so that a magnified image of the mounted sample can be
displayed on a video monitor. Cross-hairs on the video display
indicate the desired position of the sample, corresponding to the
intersection of the center of the X-ray beam with the axis of the
spindle. In order to maintain the sample at a sufficiently low
temperature once it is mounted, a stream of cold nitrogen gas is
directed at the sample mount position.
[0007] The procedure for mounting and aligning a sample manually is
described below. An operator places a sample into a small canister
of liquid nitrogen and then maneuvers the canister near to the
sample mount position on the head of the positioning device. As
quickly as possible, the operator withdraws the sample and mounts
it onto the head of the positioning device. Using the video image
on the monitor, the operator turns adjustment screws controlling
the "X", "Y", and "Z" axes until the sample is centered within the
X-ray beam and spindle axes (as indicated by the cross-hairs on the
video display). After the sample has been centered, analysis of the
sample by X-ray diffraction is begun. The procedure is described in
detail in Garman, et al., "Macromolecular Cryocrystallography", J.
Appl. Cryst. (1997) 30, 211-237 (hereinafter "Garman et al."),
incorporated herein by reference.
[0008] According to Garman et al., there are numerous problems
involved in manual procedures for X-ray-diffraction data collection
from macromolecular crystals at cryogenic temperatures. According
to Garman, prerequisites for starting a cryogenic data collection
are a reliable cryostat, the ability to maintain an ice-free
environment, some crystal-mounting equipment, a sufficient number
of crystals, and some manual dexterity for smooth and rapid
operation on the part of the operator. An important part of a
cryocrystallographic data collection is the method of crystal
mounting and the hardware associated with it. Macromolecular
crystals require special treatment compared to crystals of small
molecules, because macromolecular crystals have a liquid content
ranging from approximately 5 to 70%. The current most widely used
technique is the loop method, wherein a loop is used to suspend a
crystal by surface tension in a thin film of cryoprotected buffer.
The first loops were made of gold-plated tungsten wire. These metal
loops were replaced by loops made from various fine (10-50 .mu.m
diameter) fibers that do not absorb and scatter X-rays to the same
extent as metal, such as hair, fibers of glass, nylon, rayon,
fly-fishing threads, unwaxed dental floss, cotton, surgical thread
and mohair wool.
[0009] There are several ways of connecting the loop-supporting pin
to the head of the positioning device. Two widely used methods are
insertion of a pin directly into the hole in the head of the
positioning device and attachment of a magnet to the head of the
positioning device, to which a magnetic pin-holder is attracted and
rigidly held.
[0010] Evaporation from the film suspended in the loop is very
rapid because of its large surface-to-volume ratio. Therefore, one
of the most critical parameters in a cryocrystallographic
experiment is the time between picking up the crystal and flash
cooling it. This time should be as short as possible, ideally less
than one second, otherwise the crystal can dehydrate or components
of the buffer can precipitate. According to Garman et al., all
manipulations and motions should be practised on several dry runs
with nothing in the loop, to ensure smooth and rapid operation
later on. No time should be wasted in viewing the crystal within
the loop, since flash cooling an empty loop is less harmful than
losing crystals before cooling by stopping to check whether they
really are in the loop.
[0011] For most protein crystals, flash cooling in a gas stream is
perfectly adequate and represents the safest and simplest option.
From a practical standpoint, for gas-stream flash cooling it is
helpful at first to have a second operator present who can divert
the cold gas stream by holding a piece of card over it as soon as
the "fisher" signals that the crystal is caught. Once the crystal
is positioned, the card is then swiftly whipped away ensuring rapid
and reproducible cooling. Experienced cryocrystallographers tend to
divert the cold gas stream themselves or do not divert it at all
while placing the crystal on the head of the positioning device,
success depending on the quickness and certainty of their
action.
[0012] The most common difficulty experienced by experimenters
starting to use cryotechniques is ice around, near, on, and/or in
the crystal. There are several reasons for ice forming around the
crystal. The end of the cryonozzle may be positioned too far from
the crystal: ideally it should be as close as possible since the
temperature profile of the cold nitrogen stream is very sharp (the
temperature rises from 100.degree. K to room temperature over a few
millimeters for most open-flow systems). In addition, further away
from the nozzle the gas stream becomes dissipated and is thus more
susceptible to the effects of turbulence and drafts. If placing the
cryonozzle near the crystal results in a shadow on the X-ray
detector, thought should be given to changing the angle of approach
of the stream. If this proves impossible, the shadow can be masked
out during data processing.
[0013] A question that often arises concerns the optimum angle of
incidence of the cold stream on the crystal. This is not an
important factor in a draft-free and carefully monitored
experiment. However, most cold streams operate better with the gas
flowing downwards. Also, experimental constraints must be taken
into account. For instance, for crystal storage enough space must
be available to allow cryovial access.
[0014] In general, a major reason for ice formation is turbulent
flow at the boundaries between the cold gas and warm coaxial stream
and between the latter and warm wet air in the room. To prevent
this and to allow the desired laminar flow, the flow velocities of
the cold and the warm dry gases must be matched. To match the
flows, the relative areas of the two gas streams can be calculated
and the rates scaled accordingly.
[0015] Many classes of biological molecules can be studied by X-ray
crystallography, including, but not limited to, proteins, DNA, RNA,
and viruses. Scientists have reported the crystal structures of
molecules that carry ligands within their receptors, i. e.,
ligand-receptor complexes.
[0016] Given a representation of a target molecule or
ligand-receptor complex, scientists can search for pockets or
receptors where biological activity can take place. Then scientists
can experimentally or computationally design high-affinity ligands
(or drugs) for the receptors. Computational methods have
alternatively been used to screen for the binding of small
molecules. However, these previous attempts have met with limited
success. Several problems plague ligand design by computational
methods. Computational methods are based on estimates rather than
on exact determinations of the binding energies, and rely on simple
calculations when compared with the complex interactions that exist
within a biomolecule. Moreover, computational models require
experimental confirmation, which often expose the models as false
positives that do not work on the actual target.
[0017] It has recently been discovered that X-ray crystallography
can be used to screen compounds that are not known ligands of a
target biomolecule for their ability to bind the target. The method
comprises the steps of obtaining a crystal of a target biomolecule,
exposing the target to one or more test samples that are potential
ligands of the target, and determining whether a ligand/biomolecule
complex is formed. The target is exposed to potential ligands by
various methods, including but not limited to, soaking a crystal in
a solution of one or more potential ligands or co-crystallizing a
biomolecule in the presence of one or more potential ligands.
Structural information from the ligand-receptor complexes found can
be used to design new ligands that bind tighter, bind more
specifically, have better biological activity or have better safety
profiles than known ligands.
[0018] According to this novel method, ligands for a target
molecule having a crystalline form are identified by exposing a
library of small molecules, either singly or in mixtures, to the
target (e. g., protein, nucleic acid, etc.). Then, one obtains
crystallographic data to compare the electron density map of the
putative target-ligand complex with the electron density map of the
target biomolecule. The electron density map simultaneously
provides direct evidence of ligand binding, identification of the
bound ligand, and the detailed three-dimensional structure of the
ligand-target complex. Binding may also be monitored by changes in
individual reflections within the crystallographic diffraction
pattern which are known to be sensitive to ligand binding at the
active site. This could serve as a pre-screen but would not be the
primary method of choice because it provides less detailed
structural information.
[0019] By observing changes in the level of ligand electron density
or the intensity of certain reflections in the diffraction pattern
as a function of ligand concentration either added to the crystal
or in co-crystallization, one may also determine the binding
affinities of ligands for biomolecules. Binding affinities may also
be obtained by competition experiments. Here, the new compound(s)
are soaked or co-crystallized with one of a series of
diversely-shaped ligands of known binding affinity. If the known
ligand appears in the electron density map, the unknown ligands are
weaker binders. However, if one of the new compounds is found to
compete for the site, it would be the tighter binder. By varying
the concentration or identity of the known ligand, a binding
constant for the hit may be estimated.
[0020] Screening requires exposing a target molecule to thousands
of compounds singly or in mixtures. Screening by means of X-ray
crystallography requires examining many crystals, which in turn can
involve many days of operating 24 hour per day. Such thorough
screening can only be accomplished by means of an automated system
for mounting crystals onto the X-ray instrument and for aligning
the crystals to the X-ray beam.
[0021] The use of cryogenic techniques brings great advantages to
the crystallographer. One advantage is that the great reduction in
radiation damage to crystals at cryogenic temperatures gives the
crystallographer effectively infinite crystal lifetimes on an
in-house source and vastly extended lifetimes on a synchrotron.
Another advantage of cryogenic data collection is that the
crystal-mounting methods used are mechanically gentler and involve
less sample handling. A third advantage of the technique is the
facility for in-house screening of flash-cooled crystals and the
possibility of storing and transporting them.
[0022] The major problem with the use of cryogenic techniques is
the high expense of trained operators to mount the samples and
collect the data. Therefore, it would be desirable to develop a
method for collecting X-ray crystallographic data automatically,
without the necessity of a trained operator being present.
[0023] Synchrotron X-radiation has become a very common source of
X-rays for examining crystals of all types of molecules, small and
macromolecular. Because of the particularly intense X-rays
available at a synchrotron, cryocooling of samples is usually
desirable and often necessary. Although the intense X-rays result
in a large reduction in data collection times, often as low as
minutes, safety issues complicate sample loading so that the steps
of crystal mounting and alignment to the X-ray beam often take as
long as or even longer than the data collection step itself. The
duration of these mounting and alignment steps results in a
significant lowering of efficiency in the use of synchrotron
beamlines, which are in great demand and expensive to construct and
operate. An automated device for mounting crystal samples onto the
X-ray instrument and for accurately aligning the crystal samples to
the X-ray beam would significantly reduce the time required for
sample loading and greatly accelerate the process of examining a
great number of samples. Thus, an automated device would achieve a
significant increase in efficiency in the use of synchrotron
beamlines.
SUMMARY OF THE INVENTION
[0024] In one aspect, this invention provides a method for mounting
a sample comprising a crystal for X-ray crystallographic analysis,
which method comprises the steps of:
[0025] (a) providing a crystal holder containing at least a
crystal;
[0026] (b) providing a tool capable of retrieving the crystal
holder, the tool movable by means of a robot;
[0027] (c) providing a positioning device for mounting the crystal
holder so that the crystal is in the path of a beam of X-rays;
and
[0028] (d) activating the robot so that the tool retrieves the
crystal holder, transfers the retrieved crystal holder to the
positioning device, and mounts the transferred crystal holder on
the positioning device.
[0029] In another aspect, this invention provides a method for
aligning a sample comprising a crystal for X-ray crystallographic
analysis, which sample is mounted on a positioning device. The
method comprises the steps of:
[0030] (a) providing a sample, the sample mounted on a positioning
device;
[0031] (b) providing an apparatus capable of viewing the mounted
sample, whereby the apparatus is capable of imaging said mounted
sample and determining coordinates of the sample relative to a
reference position;
[0032] (c) providing a source of power for adjusting the
positioning device linearly along three orthogonal axes and
rotationally about one of the three axes; and
[0033] (d) activating the source of power to cause the positioning
device to be adjusted such that the sample is positioned into the
path of a beam of X-rays, the adjustments of the positioning device
being at a plurality of angles, such that the sample is positioned
within the beam of X-rays at any angle of rotational
adjustment.
[0034] In still another aspect, this invention provides a method
for determining the structure of a sample containing a crystal by
means of X-ray crystallography, which method comprises the steps
of:
[0035] (a) providing a crystal holder containing at least a
crystal;
[0036] (b) providing a tool capable of retrieving the crystal
holder, the tool movable by means of a robot;
[0037] (c) providing a positioning device for mounting the crystal
holder so that the crystal is in the path of a beam of X-rays;
[0038] (d) activating the robot so that the tool retrieves the
crystal holder, transfers the retrieved crystal holder to the
positioning device, and mounts the transferred crystal holder on
the positioning device;
[0039] (e) providing an apparatus capable of viewing the mounted
sample, whereby the apparatus is capable of imaging said mounted
sample and determining coordinates of the sample relative to a
reference position;
[0040] (f) providing a source of power for adjusting the
positioning device linearly along three orthogonal axes and
rotationally about one of the three axes;
[0041] (g) activating the source of power to cause the positioning
device to be adjusted such that the sample is positioned into the
path of a beam of X-rays, the adjustment of the positioning device
being at a plurality of angles, such that the sample is positioned
within the beam of X-rays at any angle of rotational
adjustment;
[0042] (h) providing a beam of X-rays, the beam aimed at the
sample; and
[0043] (i) recording scattering of X-rays from the sample.
[0044] In still another aspect, this invention provides a device
for holding a crystal comprising:
[0045] (a) a base;
[0046] (b) an attachment element projecting from the base;
[0047] (c) a stem projecting from the attachment element, the stem
supporting a loop for holding the crystal; and
[0048] (d) at least one aperture in the attachment element for
allowing venting of the device, the device capable of being
attached to both a storage cell and a positioning device.
[0049] In still another aspect, this invention provides an
apparatus for retrieving a crystal holder from a storage cell
comprising:
[0050] (a) a rotatable element capable of retrieving the crystal
holder from the storage cell;
[0051] (b) a means for rotating a rotatable element in a given
direction of rotation when the rotating means is in a locked
mode;
[0052] (c) a means for providing a controlled amount of torque when
the rotating means is slipping relative to the rotatable element;
and
[0053] (d) a means for activating the rotating means and the torque
controlling means.
[0054] In a preferred embodiment, an apparatus for retrieving the
crystal holder from the storage cell comprises:
[0055] (a) a clutch having a cylindrical housing, the housing
comprising a bore surrounded by a wall;
[0056] (b) a cylindrical plunger capable of moving axially within
the bore of the housing;
[0057] (c) the plunger having at least one elongated grove on the
exterior periphery thereof, the groove capable of receiving a
locking pin;
[0058] (d) the housing having at least one aperture extending
through the wall thereof;
[0059] (e) at least one spring pin retained in the aperture, the
pin capable of engaging the elongated groove when the plunger is
disposed in a first position in the housing, the pin capable of
disengaging the elongated groove when the plunger is disposed in a
second position in the housing;
[0060] (f) a means in the housing for resiliently biasing the
plunger toward the first position in the housing;
[0061] (g) a friction plate in contact with the interior wall of
the housing, the friction plate providing friction between an
output flange and the friction plate; and
[0062] (h) a shaft attached to the plunger, the shaft capable of
transmitting torque to the friction plate, the shaft further
capable of moving axially with respect to the friction plate.
[0063] In still another aspect, this invention provides a device
for holding a plurality of samples, the device comprising a
plurality of storage cells. The device is capable of maintaining
the temperature of the samples at a temperature of not greater than
about 160.degree. K. Each of the storage cells has a guided
passageway; the guided passageway has a base at the lower end
thereof and an opening at the upper end thereof. The area of the
opening is greater than the area of the base. At least one
side-wall circumscribes the base and the opening. The base is of
sufficient area to allow placement of a sample holder. The opening
is of sufficient area to allow ingress of a tool for retrieving the
sample holder. The base has attached thereto a means for locking
the sample holder to the sample-holding device. The device may also
be equipped with a lid that can be moved by means of a robot.
[0064] This invention also provides various tools and auxiliary
devices for carrying out the methods described herein.
[0065] This invention provides numerous advantages over
conventional methods of X-ray crystallography. First, this
invention makes it possible to reduce the number of trained
operators required to conduct X-ray analysis of crystals. Second,
this invention makes it possible to analyze crystals without the
need for an operator to be present. Third, this invention makes it
possible to increase the speed of analysis by X-ray
crystallography, thereby increasing the throughout of the analysis.
Fourth, this invention makes it possible to standardize the
handling of samples and reduce the possibility of errors by the
operator. This invention also facilitates collection of data 24
hours per day, seven days per week, thereby increasing the
utilization of expensive X-ray crystallography equipment. This
invention further facilitates the retrieval and preservation of
crystal samples after data has been collected, thereby making it
possible to re-analyze the sample at a later date.
BRIEF DESCRIPTION OF THE DRAWINGS
[0066] FIG. 1 is a schematic diagram depicting a system for
performing the method of this invention.
[0067] FIG. 2 is a plan view of a sample rack of this
invention.
[0068] FIG. 3 is a view in cross-section taken along line 3-3 of
the sample rack of FIG. 2.
[0069] FIG. 4 is a view in cross-section of a magnetic base at the
lower end of a storage cell to which can be attached a crystal
holder.
[0070] FIG. 5 is a side view in elevation of a crystal holder.
[0071] FIG. 6 is a top plan view of a crystal holder.
[0072] FIG. 7 is a schematic diagram in cross-section of a robot
tool for retrieving a crystal holder.
[0073] FIG. 8 is a schematic diagram in cross-section of a robot
tool having a crystal holder attached thereto.
[0074] FIG. 9 is a schematic diagram of a robot tool, clutch,
robot, and crystal holder.
[0075] FIG. 10 is a schematic diagram in cross-section of a clutch
assembly for transmitting torque to the robot tool. In this figure,
the clutch is in a locked mode.
[0076] FIG. 11 is a schematic diagram in cross-section of a clutch
assembly for transmitting torque to the robot tool. In this figure,
the clutch is in a slipping mode.
[0077] FIG. 12 is a plan view of a magnetic base for the end of the
positioning device of the system.
[0078] FIG. 13 is a side view in elevation of the magnetic base of
FIG. 12.
[0079] FIG. 14 is a side view in elevation of a dryer for
preventing moisture from collecting on the robot tool.
[0080] FIGS. 15, 16, 17, 18, 19, 20, and 21 are photographs
illustrating the displays of the crystal at various points in the
procedure of this invention.
DETAILED DESCRIPTION
[0081] As used herein, the term "robot" means a machine or device
that works automatically or by remote control. As used herein, the
term "crystal" means an ordered array of molecules that is capable
of diffracting X-rays. As used herein, the term "sample" refers to
the crystal contained in the loop of the device for holding a
crystal.
[0082] Referring now to FIG. 1, a system 10 for carrying out the
method of this invention comprises an X-ray generator (not shown),
a positioning device, such as, for example, a goniometer, 12
mounted on a rotating spindle 14, an instrument base 16, a CCD
camera 18, an insulated container 20, a sample rack 22, a
multi-axis robot 24, a controller 26 for the robot 24, an
automation computer 28, a data collection computer 30, at least one
motor controller 32, a detector 34, a cold stream nozzle 36, a cold
stream actuator 38, and a motor 40 for translating the spindle 14
along its major axis. Although the computers 28 and 30 are shown as
individual components, they can be combined into a single unit. The
system 10 preferably also includes an automatic system 41 for
replenishing liquid nitrogen to the insulated container 20. Such
replenishing systems are well-known to those skilled in the art of
X-ray crystallography. An overview of the components for
diffractometer systems for X-ray crystallography can be found in
Kirk-Othmer Encyclopedia of Chemical Technology, Fourth Edition,
Volume 25, John Wiley & Sons (New York:1998), pages 742-759,
incorporated herein by reference. The foregoing article describes
various components, such as X-ray tubes, detectors, goniometers,
and other components typically associated with X-ray
crystallography.
[0083] Referring now to FIGS. 2, 3, 4, 5, and 6, an insulated
container 20 filled with liquid nitrogen is positioned near the
X-ray generator. A sample rack 22 holding one or more crystal
samples is mounted in the insulated container 20 so that the
samples are immersed in liquid nitrogen. The sample rack 22 is used
to store crystal samples after the crystal samples have been
mounted onto a crystal holder 42. The sample rack 22 is designed to
maintain the samples under a layer of liquid nitrogen, which is
preferably at a temperature of not greater than about 160.degree.
K. The sample rack 22 is constructed to render insertion and
removal of crystal holders 42 by either a human operator or the
robot 24 more efficient. The sample rack 22 comprises an array of
storage cells 44, each storage cell 44 capable of holding one
crystal holder 42. Each storage cell 44 comprises a magnetic base
46 and a guided passageway 48 leading from an opening 50 in the
storage cell 44 at the upper end 52 of the storage cell 44 to the
magnetic base 46 at the lower end 54 of the storage cell 44. The
guided passageway 48 is circumscribed by a wall 55 running from the
upper end 52 of the storage cell 44 to the lower end 54 of the
storage cell 44. The sample rack is preferably constructed of a
metal. It is also possible to equip the sample rack with a lid (not
shown). This optional lid can be moved by means of a robot to allow
access to the crystals holder 42 in the storage cells 44.
[0084] The guided passageway 48 is preferably constructed such that
the opening 50 at the upper end 52 of the storage cell 44 has a
greater area than the base 46 at the lower end 54 of the storage
cell 44. This type of construction makes it easy to introduce the
crystal holder 42 into the storage cell 44.
[0085] The purpose of the magnetic base 46 is to retain the crystal
holder 42 at the lower end 54 of the storage cell 44 by magnetic
attraction once the crystal holder 42 has been inserted into the
storage cell 44 by the robot 24 or the human operator. The crystal
holder 42 is preferably made of a material that is magnetically
attracted to a ferromagnetic material. A pin 56 extending radially
outward from a ferromagnetic material 58 of the magnetic base 46
engages a notch 60 in the base 62 of the crystal holder 42 to
prevent relative rotation between the crystal holder 42 and the
magnetic base 46. The guided passageway 48 guides the movement of
the human operator or the robot 24 when the crystal holder 42 is
inserted into the storage cell 44. In addition, the guided
passageway 48 protects neighboring samples from damage when a human
operator is inserting or removing a crystal holder 42. This feature
is especially important in the case of a human operator, because
visibility is limited when the sample rack 22 is filled with liquid
nitrogen.
[0086] The crystal holders 42 are preferably fabricated from a
ferromagnetic material, such as, for example, steel. The use of a
ferromagnetic material allows secure attachment of the crystal
holder 42 to the magnetic mount 63 that is attached to the end 64
of the positioning device 12 and the magnetic base 46 of the
storage cell 44 in the sample rack 22. An attachment element 66 of
the crystal holder 42 projects from the base 62 of the crystal
holder 42. The attachment element 66 is threaded with a standard
male screw thread 67. The attachment element 66 can be attached to
a mating female screw thread 68 incorporated into an end 70 of a
robot tool 72. Such a screw means of attachment is preferred,
because it provides an efficient retrieval method for the samples
when they are immersed in liquid nitrogen. Projecting from the
attachment element 66 of the crystal holder 42 is a stem 74, at the
end of which is located a loop 76 for holding the crystal sample.
The attachment element 66 preferably has an aperture 77 formed
therein to allow liquid nitrogen to flow through the crystal holder
42.
[0087] The multi-axis robot 24 is mounted near the insulated
container 20 and within reach of the positioning device 12.
Referring now to FIGS. 7 and 8, the robot 24 has an extension
referred to herein as the robot tool 72, which contains a female
screw thread 68 for mating with the male screw thread 67 of a
crystal holder 42. The robot tool 72 is capable of retrieving the
crystal holder 42, which contains the sample, from the sample rack
22 and inserting the crystal holder 42 onto the positioning device
12 for data collection. The robot tool is also capable of
retrieving the crystal holder 42 from the positioning device 12 and
inserting the crystal holder 42 into the sample rack 22 for
storage. The robot tool 72 is designed to maintain the sample at a
cryogenic temperature, near to that of liquid nitrogen, i.e., a
temperature not in excess of about 160.degree. K., during the short
time that the sample is in transit. The robot tool 72 is also
designed to shield the sample from ambient air when the crystal
holder 42 is united with the robot tool 72. The robot 24 is able to
retrieve the crystal holder 42 by contacting the crystal holder 42
with the end 70 of the robot tool 72 and rotating the robot tool 72
clockwise so that the crystal holder 42 and the robot tool 72 are
screwed together.
[0088] Before being used to grasp the crystal holder 42, the robot
tool 72 is immersed in liquid nitrogen for a short period of time
(typically 20 seconds) in order to cool the robot tool 72 to a
temperature near to that of liquid nitrogen. Vent apertures 80 in
the body 82 of the robot tool 72 allow air to escape the interior
cavity 84 of the robot tool 72 as liquid nitrogen fills the
interior cavity 84 of the robot tool 72. When the robot tool 72 and
the crystal holder 42 are joined, the crystal sample is maintained
at a low temperature by the liquid nitrogen inside the interior
cavity 84 and by the cold metal walls 86 surrounding the interior
cavity 84 of the robot tool 72. Liquid nitrogen flows through the
aperture 77 in the crystal holder 42 into the interior cavity 84 of
the robot tool 72 when the robot tool 72 and the crystal holder 42
attached thereto are immersed in liquid nitrogen.
[0089] The positioning device 12, which is translatable along an
X-axis and a Y-axis via stepper motors 88 and 90, respectively, is
mounted onto a rotating spindle 14 on the X-ray diffraction
instrument. The positioning device 12 is also translatable along a
Z-axis by means of a stepper motor 40. Home position sensors (not
shown) are built into the X-axis, Y-axis, and Z-axis translations
so that a reference position can be found at any time. The stepper
motors and the home sensors for the X-axis, Y-axis, and Z-axis
translations are connected to a motor controller 32, which in turn
communicates with an automation computer 28. Rotation of the
spindle 14 is controlled in a similar manner by commands
communicated by the automation computer 28. Motions of the
multi-axis robot 24 are controlled by the robot controller 26,
which also communicates with the automation computer 28. Video
output from the CCD camera 18 is input into a frame grabber video
card (not shown), which is connected with the automation computer
28. A communication connection is provided between the automation
computer 28 and a separate data collection computer 30. Operation
of the automated system is described below.
Operation
[0090] The operation of this invention involves retrieving samples,
contained in crystal holders, from a storage area, mounting the
retrieved samples on a positioning device, aligning the mounted
samples prior to collecting data, collecting data, and returning
the sample to the storage area.
[0091] An operator "enters" the identification numbers of the
samples that are to be analyzed and initiates the automated process
by entering an appropriate command into the data collection
computer 30. After this point, no operator intervention is
required.
[0092] A stored program within the robot controller 26 is activated
and the robot 24 retrieves a sample from the sample rack 22 located
in the insulated container 20. The robot tool 72 enables the robot
24 to grip the sample while the sample is immersed in liquid
nitrogen. The sample is then withdrawn from the sample rack 22 and
immediately installed on the magnetic mount 63 on the end 64 of the
positioning device 12.
[0093] In order for the robot tool 72 to reliably grasp and release
the sample, which is disposed in the crystal holder 42, a clutch 94
connected between the rotation stage 96 of the robot 24 and the
robot tool 72 is used. The clutch 94 is illustrated in detail in
FIGS. 10 and 11. In general, the clutch can have any of numerous
configurations, but, at minimum, in a generic sense, the clutch 94
comprises:
[0094] (a) a rotatable element capable of retrieving the crystal
holder from the storage cell;
[0095] (b) a means for rotating the rotatable element in a given
direction of rotation when the rotating means is in a locked
mode;
[0096] (c) a means for providing a controlled amount of torque when
the rotating means is in a slipping mode relative to the rotatable
element; and
[0097] (d) a means for activating the rotating means and the torque
controlling means.
[0098] The clutch 94 is normally in the locked mode. When the
clutch 94 is in the locked mode, rotation initiated at an input
flange 98 is directly transmitted to an output flange 100 without
allowing rotational slippage. Spring pins 102 protrude into axial
grooves 104 in a plunger 106, which is disposed in the bore of a
housing 108, thereby preventing rotation of the plunger 106 in the
housing 108. Preferably, the plunger 106 and the housing 108 are
cylindrical in shape. When the clutch 94 is in the locked mode, any
amount of torque can be transmitted to the robot tool 72, up to the
torque limits of the robot 24 itself.
[0099] To switch the clutch 94 to the slipping mode, an axial force
must be imposed on the clutch 94 so that the plunger 106 is shifted
relative to the housing 108, typically by approximately 0.2 inches
to the left. When the plunger 106 is shifted, the spring pins 102
are disengaged from the grooves 104 in the plunger 106, thereby
allowing relative rotation between the plunger 106 and housing 108,
and thus between the input flange 98 and the output flange 100.
However, when relative rotation occurs, a controlled amount of
rotational friction is generated by a friction plate 110, which
includes a plate 112 and an o-ring 114, as the friction plate rubs
against the output flange 100. A spring 116 resiliently biases the
friction plate 110 toward the output flange 100. The level of
friction between the friction plate 110 and the output flange 100
can be controlled by appropriate selection of the material and the
properties of the o-ring 114 and the spring 116. Materials and
properties for the plate 112, o-ring 114, and spring 116 are
matters of design choice, and appropriate selection thereof is
well-known to those of ordinary skill in the art.
[0100] The clutch 94 operates in accordance with the following
procedure:
[0101] (1) The crystal holder 42, which holds the sample, is seated
in the sample rack 22, on the magnetic base 46 of the storage cell
44. The sample rack 22 is immersed in a container 20 of liquid
nitrogen.
[0102] (2) The robot 24 points the robot tool 72 downwardly and
moves to a position above the storage cell 44 in the sample rack 22
near the desired crystal holder 42.
[0103] (3) The robot 24 moves the robot tool 72 downwardly until
the robot tool 72 just contacts the crystal holder 42. The robot 24
pauses in this position in order to allow the robot tool 72 to cool
to a temperature near that of liquid nitrogen. The vent apertures
80 in the robot tool 72 allow the interior cavity 84 of the robot
tool 72 to become filled with liquid nitrogen.
[0104] (4) The robot 24 moves the robot tool 72 downwardly about
0.25 inch in order to apply axial force to the clutch 94 and to
unlock the clutch 94.
[0105] (5) Through the use of the rotation stage 96, the robot 24
rotates the robot tool 72 clockwise to screw the crystal holder 42
onto the robot tool 72. The threads 67 of the crystal holder 42
unite with the threads 68 of the robot tool 72. Generally, only
approximately one full turn of the robot tool 72 is required to
fully screw the crystal holder 42 onto the robot tool 72.
Preferably, two additional turns of the robot tool 72 are made to
ensure that the crystal holder 42 is fully engaged on the robot
tool 72. Because the clutch 94 is unlocked, rotation-wise slippage
will occur after the crystal holder 42 becomes fully engaged (i.e.,
fully screwed on to the robot tool 72). If the clutch 94 were
unable to slip, breakage or robot overload would likely occur after
full engagement of the crystal holder 42 and the robot tool 72.
Axial compliance of the clutch 94, coupled with its ability to slip
when unlocked, provides a degree of "forgiveness" in the system.
The movements of the robot 24 do not have to match the position or
thread length of the crystal holder 42 perfectly. Small errors in
robot movements and programming are tolerated because of the axial
compliance and slippage of the clutch 94.
[0106] (6) After the crystal holder 42 is fully engaged by the
robot tool 72, the robot 24 withdraws the robot tool 72 with
crystal holder 42 from the sample rack 22 and performs the next
operation. The crystal sample on the crystal holder 42 is protected
from the warm atmosphere, because it is surrounded by liquid
nitrogen inside the interior cavity 84 of the robot tool 72 and
protected by the cold metal walls 86 of the robot tool 72
itself.
[0107] The robot tool 72 then mounts the crystal holder 42 onto the
positioning device 12 in the following manner.
[0108] (1) the robot 24 guides the robot tool 72 to a horizontal
position (parallel to the base 16) and moves the robot tool 72 to a
position near the end 64 of the positioning device 12.
[0109] (2) The robot 24 moves the robot tool 72 toward the magnetic
mount 63 until the crystal holder 42 just contacts the magnetic
mount 63 at the end 64 of the positioning device 12. The angular
position of the crystal holder 42 is such that the notch 60 in the
base 62 of the crystal holder 42 engages an alignment pin 118 of
the magnetic mount 63. This engagement prevents angular rotation of
the crystal holder 42 relative to the magnetic mount 63. As shown
in FIGS. 12 and 13, the magnetic mount 63 also includes a magnet,
i. e., a ferromagnetic material, 120 and an attachment pin 122. The
magnet 120 serves to retain the crystal holder 42 by magnetic
attraction after the crystal holder 42 has been mounted onto the
magnetic mount 63 on the end 64 of the positioning device 12. The
attachment pin 122 serves to attach the magnetic mount 63 to the
end 64 of the positioning device 12. At this point, the clutch 94
is locked because it has not been significantly compressed in the
axial direction.
[0110] (3) The robot 24 rotates the robot tool 72, via the rotation
stage 96, in the counter-clockwise direction, preferably two turns,
to ensure that the robot tool 72 is completely unscrewed from the
crystal holder 42. Because the clutch 94 is locked, sufficient
torque can be applied to unscrew the crystal holder 42 from the
robot tool 72, even if the crystal holder 42 and the robot tool 72
are stuck or frozen together.
[0111] (4) While rotating in the counter-clockwise direction, the
robot tool 72 is drawn away from the end 64 of the positioning
device 12, thereby leaving the crystal holder 42 adhered to the
magnetic mount 63 at the end 64 of the positioning device 12.
[0112] After the crystal holder 42 is mounted onto the positioning
device 12, the crystal holder 42 must be properly positioned for
data collection. Prior to positioning the crystal holder 42, the
robot tool 72 is quickly moved away from the positioning device 12
to a "rest" position in a dryer 124. The dryer 124 is shown in FIG.
14. The purpose of the dryer 124 is to prevent moisture from
collecting on the robot tool 72 when the robot tool 72 is not in
use. A stream of dry gas, e. g., nitrogen, at ambient temperature,
is introduced at port 126, traverses an interior chamber 128, and
exits at port 130. The material of construction of the dryer is not
critical. When the robot tool 72 is inserted into the interior
chamber 128 of the dryer 124, the dry gas prevents moisture from
collecting on the robot tool 72.
[0113] After being mounted on the positioning device 12, the
temperature of the sample is maintained at a low temperature by a
cold stream, which is provided through the cold stream nozzle 36,
which is positioned as close to the sample as possible. The cold
stream nozzle 36 is mounted onto the cold stream actuator 38, so
that the cold stream nozzle 36 can be retracted when the crystal
holder 42 is mounted onto the positioning device 12 and extended at
other times.
[0114] At this time, an image processing/sample alignment program
is employed to automatically position the sample at the
intersection of the X-ray beam and the axis of the spindle. The
alignment procedure technique uses a "machine vision" algorithm to
analyze the video information obtained via the CCD camera 18
mounted in the base 16 of the system 10. The alignment procedure
repeatedly invokes the machine vision algorithm as described below,
and uses the position information obtained to reposition the sample
by means of the stepper motors 88, 90, and 40. The cycle described
below is repeated until the difference between the actual sample
position and the desired sample position is sufficiently small for
the purpose of data collection.
[0115] The details of a "machine vision" algorithm suitable for
this invention will now be discussed. The "machine vision"
algorithm can find the centroid and the "tip" (leftmost point in
the image) of the crystal sample. In the following discussion, the
Z-axis is the axis of rotation of the sample. The .phi. angle is
the angle of rotation about the Z-axis. The X-axis is the axis
horizontal to the instrument base 16 (and CCD camera 18) when the
.phi. angle is 0.degree., and the Y-axis is the other orthogonal
axis. When the .phi. angle is 0.degree., the vertical direction of
the image corresponds to the X-axis while the horizontal direction
of the image corresponds to the Z-axis. When the .phi. angle is
90.degree., the vertical direction of the image corresponds to the
Y-axis while the horizontal image still corresponds to the
Z-axis.
[0116] The machine vision algorithm begins with a digitized image,
represented as a matrix of eight bit numbers corresponding to the
pixels in the image. A typical starting image is shown in FIG. 17.
The goal of the image processing method described below is to
determine the centroid of the loop 76 of the crystal holder 42 as
shown in the center of FIG. 17. The machine vision algorithm must
be capable of discriminating between the loop 76 of the crystal
holder 42 and the other elements of the image. These elements
include the crosshairs and reticle graduations and the stem 74 of
the crystal holder 42. At a minimum, the machine vision algorithm
comprises the following steps:
[0117] (1) Ignore the "rightmost 10% of the image as shown in FIG.
18.
[0118] (2) Convert the eight bit grayscale image to two bit black
and white image by converting the darkest 12% of the pixels to
black and the remaining pixels to white as shown in FIG. 19.
[0119] (3) Blank the leftmost 20%, the topmost 10%, and the
bottommost !0% of the image, thereby eliminating the effect of the
shadow of the cold stream and reducing the influence of non-uniform
illumination, as shown in FIG. 20.
[0120] (4) Perform a "thinning" algorithm as follows:
[0121] (a) for each dark pixel, a 20.times.20 window (with the dark
pixel at the center of the window) is examined;
[0122] (b) if fewer than 280 dark pixels are contained in this
window, the pixel is changed to white;
[0123] (c) this eliminates grid lines and other artifacts, as shown
in FIG. 21.
[0124] (5) Calculate the centroid of the remaining black
pixels.
[0125] Various refinements of the foregoing algorithm have been
developed. These refinements utilize techniques known to those
skilled in the art. These refinements increase reliability for
unusual cases, such as the case of very small crystals.
[0126] The alignment procedure consists of two parts--an initial
acquisition phase followed by a fine centering phase. In the
initial acquisition phase, the sample is moved by the motors along
the X-axis and the Z-axis to a starting position. The starting
position is defined such that the system will know that the sample
is either out of the camera image completely or in the right half
of the camera image. It is important to begin in this position so
that the stem 74 on which the sample loop 76 is mounted will not
confuse the system.
[0127] The "machine vision" algorithm is then invoked. If the
sample is not found, a search pattern commences. The search pattern
involves a zigzag motion of the sample along the X-axis and the
Z-axis, invoking the machine vision algorithm at each position in
the search pattern. Once the sample is found, the system uses the
centroid information obtained from the machine vision algorithm to
center the sample to the X-axis and the Z-axis, by means of stepper
motors 88 and 40. The centering is then repeated to account for the
possibility that the sample was not completely within the field of
view of the CCD camera 18. This step completes the initial
acquisition phase of the alignment routine. At this point, the
position of the sample along the X-axis and the Z-axis should be
reasonably close to proper alignment, but the Y-axis will typically
be mis-aligned by a significant amount. In theory, all that should
have to be done to complete the alignment is to move the .phi.
angle to 90.degree. and repeat the centering process. However,
there is a potential problem with this technique for some samples.
Many crystal samples are very flat. When the face of the crystal is
viewed, the "machine vision" algorithm is very accurate (see FIG.
15). However, when the edge of the crystal is viewed, it is
difficult to distinguish between the sample, the loop 76, and the
stem 74 of the loop 76. An example of this problem can be seen in
FIG. 16. Thus, attempting to properly align the sample by using
only two angles (0.degree. and 90.degree.) is potentially harmful
to accuracy if one of the two angles happens to result in a
"machine vision" image similar to that of FIG. 16 or if the sample
has some other feature that makes it unusual, thereby confusing the
"machine vision" algorithm. To adjust for this possibility, the
system uses information at a plurality of angles between 0.degree.
and 90.degree., inclusive, to ascertain the most likely true
position of the crystal sample in three dimensions. The algorithm,
as currently implemented, is as follows:
[0128] (1) Move the .phi. angle from 0.degree. to 90.degree. in
5.degree. increments, using the "machine vision" algorithm to find
the centroid of the crystal sample at each angle.
[0129] (2) Perform a parametric least squares fit to the
equation:
V.sub.i=.DELTA.X cos .phi..sub.i+.DELTA.Y sin .phi..sub.i
[0130] where V.sub.i represents the vertical offset of the centroid
of the image of the crystal sample at the angle .phi..sub.i, and
.DELTA.X represents the unknown offset of the centroid of the image
of the sample from the X-axis and the Y-axis and .DELTA.Y
represents the unknown offset of the centroid of the image of the
sample from the Y-axis.
[0131] (3) When the values of .DELTA.X and .DELTA.Y are found,
adjust the motors to center the sample.
[0132] (4) Adjust the Z-axis by using a simple average of all of
the offsets from the horizontal in the image.
[0133] (5) Repeat the above steps (1), (2), (3), and (4) with the
exception that the .phi. angle is moved from 90.degree. to
0.degree. in 5.degree. increments.
[0134] (6) Continue to iterate the foregoing steps (1), (2), (3),
(4), and (5) until the closure criteria are met. Currently, the
closure condition is one of the following:
[0135] (a) current sum of the squares of the offsets (from the
X-axis, the Y-axis, and the Z-axis) is less than 225 pixels
squared.
[0136] (b) difference between the current sum of the squares of the
offsets and the immediately previous iteration of the sum of the
squares of the offsets is less than 225 pixels squared.
[0137] (c) six (6) iterations without fulfilling (a) or (b).
[0138] The first condition (a) is considered perfect alignment. The
second condition (b) is a case where alignment is no longer being
significantly improved, and prevents oscillation between two
equally good solutions. The final condition (c) is considered an
alignment failure.
[0139] (7) When the closure condition is met, alignment success or
failure is reported to the data collection computer 30. The data
collection computer 30 then commences taking data if the alignment
was successful, or requests the next crystal if the alignment
failed.
[0140] This technique uses information at 19 different angles;
thus, it is more robust to errors at a certain angle than an
algorithm that uses only two angles.
[0141] At this point, a signal is sent to the data collection
computer 30 and the X-ray diffraction analysis of the sample is
begun.
[0142] At the end of the data collection phase, a computer program
stored in the robot controller 26 is activated, guiding the robot
24 to retrieve the sample from the positioning device 12 and return
it to its original position in the sample rack 22.
[0143] The following procedure is employed to return the crystal
holder 42 to the sample rack 22.
[0144] (1) The robot 24 guides the robot tool 72 to a horizontal
position (parallel to the base 16) and moves the robot tool 72 to a
position near the end of the crystal holder 42. The crystal holder
42 is still mounted on the magnetic mount 63 on the end 64 of the
positioning device 12.
[0145] (2) The robot 24 moves the robot tool 72 toward the crystal
holder 42 until the robot tool 72 just contacts the crystal holder
42.
[0146] (3) The robot 24 moves the robot tool 72 about 0.2 inch
toward the positioning device 12 in order to apply axial force to
the clutch 94 and to unlock the clutch 94.
[0147] (4) By means of the rotation stage 96, the robot 24 rotates
the robot tool 72 clock-wise to screw the crystal holder 42 onto
the robot tool 72. Generally only about on full turn is required to
screw the crystal holder 42 onto the robot tool 72. Preferably, two
additional turns are made to ensure that the crystal holder 42 is
fully engaged on the robot tool 72.
[0148] (5) After the crystal holder 42 is fully engaged on the
robot tool 72, the robot 24 withdraws the robot tool and the
crystal holder 42 from the magnetic mount 63.
[0149] (6) The robot 24 points the robot tool 72 downwardly and
moves to a desired position above the appropriate storage cell 44
in the sample rack 22.
[0150] (7) The robot 24 moves robot tool 72 downwardly until the
crystal holder 42 just contacts the magnetic base 46 at the bottom
of the storage cell 44. The angular position of the crystal holder
42 is such that the notch 60 in the base 62 of the crystal holder
42 engages the pin 56 in the magnetic base 46. This engagement
prevents angular rotation of the crystal holder 42 relative to the
magnetic base 46. At this point, the clutch 94 is locked because it
has not been significantly compressed in the axial direction.
[0151] (8) The robot 24 rotates the robot tool 72 two turns in the
counter-clockwise direction to ensure that the crystal holder 42 is
fully unscrewed from the robot tool 72. Because the clutch 94 is
locked, sufficient torque can be applied to unscrew the parts, even
if the crystal holder 42 and the robot tool 72 are stuck or frozen
together.
[0152] (9) While continuing to rotate in the counter-clockwise
direction, the robot tool 72 is withdrawn from the storage cell 44
in the sample rack 22, leaving the crystal holder 42 adhered to the
magnetic base 46 at the bottom of the storage cell 44.
[0153] The entire operation process is then repeated for the next
sample to be analyzed. After all of the selected samples have been
analyzed and returned to their positions in the sample rack 22, the
robot tool 72 is parked in a rest position and the system is placed
in a standby mode.
[0154] Various modifications and alterations of this invention will
become apparent to those skilled in the art without departing from
the scope and spirit of this invention, and it should be understood
that this invention is not to be unduly limited to the illustrative
embodiments set forth herein.
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