Microdroplet High-throughput Sample Mounting Techniques in
Small Angle X-Ray Scattering
Andrew P. Huang
Office of Science, Science Undergraduate Laboratory Internship (SULI)
Washington University in St. Louis
Stanford Linear Accelerator Center
Stanford, CA
August 24, 2012
Prepared in partial fulfillment of the requirements of the Office of Science, Department of
Energy’s Science Undergraduate Laboratory Internship under the direction of Thomas M.
Weiss at the Stanford Synchrotron Radiation Lightsource (SSRL), Stanford Linear Accelerator Center.
Participant:
Signature
Research Advisor:
Signature
TABLE OF CONTENTS
Abstract
ii
Introduction
1
Basic SAXS Methodology
1
Apparatus
2
Optical Analysis
4
Results
5
Discussion and Conclusions
7
Acknowledgments
8
References
8
i
ABSTRACT
Microdroplet High-throughput Sample Mounting Techniques in Small Angle X-Ray Scattering. ANDREW P. HUANG (Washington University in St. Louis, St. Louis, MO 63105)
THOMAS M. WEISS (Stanford Synchrotron Radiation Lightsource (SSRL), Stanford Linear
Accelerator Center, Stanford, CA 94025)
Here we present a new method for automatically dispensing small amounts of protein
solution samples. The concept of the new automatic sampling device is centered about
delivering a hanging droplet into the path of an X-Ray beamline for Small Angle X-Ray
Scattering (SAXS). The sample dispensed is 3 µl or less hanging in air permitting the device
to reduce the number of windows which the beam passes through, and reduce the sampling
time. We describe in detail the methods utilized in overcoming the obstacles encountered in
the design process of such an apparatus such as maintaining droplet consistency, preventing
droplet evaporation, maintaining the overall cleanliness of the droplet dispenser system,
mitigate increased radiation damage.
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INTRODUCTION
To improve data collection efficiency and sample economy in Small Angle X-Ray Scattering
(SAXS) techniques on protein solutions, development of a new sample mounting procedure
intended to replace the current autosampler apparatus on SAXS beamline 4-2 at the Stanford Synchrotron Radiation Lightsource (SSRL) was undertaken. Currently a flow-through
capillary is used into which small aliquots (∼ 30µl) of protein (or buffer) solutions are aspirated and held into the X-ray beam for measurement. Because of imperfections in the glass
walls of the capillary tubes causing differences in scattering signal, changing capillary tubes
between measurements would cause unacceptable errors in scattering data. Therefore the
same capillary is used for the different protein and buffer solutions during an experiment
requiring extensive washing of the capillary in between scans, which takes approximately 3.5
minutes. This is a notable inefficiency especially when it is taken into consideration that the
actual scan of the sample takes approximately 30 seconds.
The basic premise behind the Microdrop Automatic Sample-mounting Technique (MAST)
is suspending a small droplet of the liquid sample (≤ 3µl) from a needle while performing
the X-ray measurement. By eliminating washing cycles the new sample mounting procedure
would allow for a substantial increase in the speed of operation at the beamline. However in
order for the new mounting procedure to be capable of matching the reliability of the current autosampler on the beamline, the issues of preventing droplet evaporation, mitigating
increased radiation damage to the sample, and maintaining droplet consistency needed to
be resolved.
BASIC SAXS METHODOLOGY
Solution based Small Angle X-Ray Scattering (SAXS) is a low resolution method for analyzing the molecular structure of proteins suspended in solution (Figure 1). Small angle
1
scattering is observed from the secondary wavelets produced by X-ray waves interacting with
individual atoms. [5] Due to the diffusion of the proteins in solution, the resolution of SAXS
is limited to the scale of a few nanometers but it can provide accurate information with
respect to size and shape of the molecules in solution. [4]
From the scattering pattern on the detector, intensity of the scattering observed is integrated radially about the center thereby yielding the SAXS scattering profile. In order
to get the scattering intensity of the protein the measured intensity of the buffer solution
must be subtracted from the scattering curve observed for the protein solution [1]. Care
in subtraction must be taken especially for intermediate to large angles as the scattering
intensity of the buffer and sample differ very slightly at these angles.
SAXS has many advantages over traditional crystallographic methods although it is of
lower resolution. Because it can be very difficult to crystallize certain proteins or often
have large quantities of a protein, SAXS permits the analysis of samples that would be
otherwise impossible to analyze by crystallography. Small angle scattering can be performed
on proteins suspended in solution to mimic certain in vivo conditions for analysis. SAXS
also permits the observation of folding of proteins in time resolved experiments and protein
behavior under different stresses.
APPARATUS
The current autosampler (see Figure 2) is designed to be a reliable sample dispenser and
buffer solutions into an X-ray beam for SAXS but unfortunately has several shortcomings
that are addressed by the next generation sample mounting procedures.The first of these are
the difficulties caused by the capillary tubes installed on the apparatus. Imperfections in the
fabrication process of the capillary tubes causes an increase in background signal lowering the
signal to noise ratio. The wash process necessary to avoid error due to differences between
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different capillary tubes takes significantly longer to complete than the actual scan of the
protein itself. Because beamtime is valuable, these inefficiencies are magnified by the large
number of samples that usually need to be scanned on a SAXS beamline per given experiment
leading to a significant amount of beamtime spent simply washing the sample stage. Also,
the current autosampler requires a large amount of sample volume for operation, which is
often difficult to acquire for certain scarce protein samples.
The modifications to the spare autosampler which ultimately became the centerpiece of
the Microdroplet Automatic Sample-mounting Technique (MAST) development project was
to remove the installed capillary tube so that when the needle is lowered the droplet would
have an open area to hang. In the sampling tube a microscope is pointed at the droplet
perpendicular to the path of the X-Ray beam as a means of optical imaging of the droplet
for droplet shape analysis.
The MAST system is controlled primarily by the SolSAXS tab of the Blu-Ice software
specifically developed for non-crystalline X-ray scattering systems [3]. The software is the
same as the control software for the current autosampler. From there, a procedure was
written utilizing commands which controlled the main XYZ triple-axis motor positioning
system (e.g. ndl up/to/down/by, ndl2cap, home) for the needle and the automatic syringe
control system (e.g. load, unload, wash). [2] In preparation for the first sample, the Tygon
tubing which connects the sample syringe and the flowcell fluid delivery system is filled with
a purified water as a working fluid to maintain precision in fluid output. A 10µl air gap is
drawn into the syringe to prevent contamination of the working fluid with sample as well as
preventing cross-contamination of samples. The general procedure developed for delivering
a sample is as follows:
1. Ndl to sample position on sample tray
2. Load 5µl into the needle
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3. Ndl2cap, move the needle into position above the X-ray beam
4. Unload 3µl into target region
The target region is protected by an evaporation shield, a small metal tube with holes for
the X-ray beam to pass through see Figure 3 label (B). Two holes which are perpendicular
to the X-Ray beam are covered with transparent windows permitting a microscope to be
placed to image the droplet. The wash process of the needle is rather quick, where:
1. The dispensed sample is taken back into the needle.
2. The needle is raised and moved to an empty sample region.
3. The used sample is dumped into an empty centrifuge tube on the sample tray.
4. The needle is raised.
5. A wash cycle of 100µl commences with 2 cycles at a rate of 50 µl/second.
6. The needle is dipped into a centrifuge tube filled with distilled water on sample tray.
7. The needle is raised.
8. A 10µ air gap is drawn into the syringe.
This procedure usually takes approximately 30 seconds to carry out and when combined
with the approximately 15 second scan time at 10 one second exposures this apparatus is
significantly faster than the current autosampler system.
OPTICAL ANALYSIS
To analyze the various aspects of droplet shape consistency and droplet evaporation an
optical method was used to make a precise measurement of the droplet position and size. A
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microscope was placed perpendicular to the X-Ray beam path to observe the shape of the
droplet, and in order to observe the maximum level of contrast (Figure 4 see droplet A) in
the droplet a fiber optic light element was pointed directly at the microscope objective [6].
From this it was gathered that the addition of ethanol, glycerol, and protein had no
significant effect on the droplet shape despite the change in surface tension. This was reflected
in the difference in path length across the droplet at a set point whose variation was smaller
than 1.3%.
RESULTS
The testing conducted for this experiment primarily utilized 10mg/ml Lysozyme as a stock
solution that was then serially diluted in a 20mM NaOAc and 150mM NaCl buffer which
had a pH ≈ 4.9. However, testing demonstrated that images taken using a concentration
series with this buffer were prone to being quickly affected with radiation damage after one
or two frames. To mitigate this problem small amounts of glycerol were added to the buffer
for testing at 2%, 5%, and 10%. The addition of glycerol seemed to have negligible effects
on the variation in shape of the droplet despite the change in surface tension so testing was
still able to continue.
One issue which affected the project from the start is the problem of droplet consistency,
where the droplets dispensed by the MAST system would have slight variations in droplet
shape as detected by the optical method. To compensate for this, a correction factor was
utilized to correct for the variation in X-Ray path length caused by shape differences.
Is (q) = I0 A(q)de−µd
(1)
The equation above illustrates that scattering intensity, Is (q), is directly proportional to
the incident beam intensity I0 , the scattering factor A(q), path length of the X-ray beam
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through the sample d, and the Beer-Lambert absorption factor e−µd . Therefore, differences
in droplet size will have a direct influence on the observed scattering intensity.
The transmitted intensity IT is related to the incident beam intensity I0 by the BeerLambert law
IT = I0 e−µd
(2)
From which one can solve for the path length, d
d=
−1 IT
ln( )
µ
I0
(3)
The intensity values, IT and I0 , are directly measured during the experiment for each
exposure, and ln( IIT0 ) can be used to correct for small differences in path length (the component
−1
µ
is a constant for the buffer and sample scans) due to variations in droplet size.
Each of the intensity values is divided by this correction factor and this reduces the error
observed in the data sets by about two times (See Figure 5).
During testing of the MAST system, a peculiar error began occurring during the dispensing of the droplet where the droplet would dispense in a skewed fashion or in extreme cases
crawl up the outside of the needle (see Figure 4 droplet C). This has since been attributed
to two factors, the first of which is a wet outside surface of the needle after the wash process. The second, is due to buildup of fluid within the small hole which used to house the
capillary tube the needle passes through which slowly occurs as sometimes the sample is not
completely drawn back into the needle before it is lifted. To remedy this several measures
were undertaken where the needle installed on the apparatus would be a special needle with
a teflon coating on the outside surface of the needle. Then after every sample, when the
needle is raised for drawing an air gap, the outside of the needle is wiped down. Finally
compressed air would be blown through the capillary hole to blow out any undesired liquid
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inside, although tubing must be raised around the dispensing area and windows prior to this
as to prevent splashing of any fluids onto the Mica windows of the vacuum chambers of the
beamline. Then the MAST apparatus is ready to accept the next sample.
The signal to noise ratio which interfered with the validity of the output data is lower for
low concentrations of proteins (See Figure 7). A comparison of the experimental Rg and the
actual Rg value range of Lysozyme 14.3 ˚
A ± 1, one can determine the lowest concentration
that will return reliable MAST results. See Table 1.
It was determined that data from protein concentrations below 2.50 mg/ml will typically
be unreliable as the average Rg here is beyond the acceptable range of error for Rg . Also
the Rg values given by the Autosampler are closer to the actual value of Rg for Lysozyme so
there is still work to be done to increase the accuracy and repeatability.
In comparison to the Autosampler technique, the MAST methodology does not seem to
return data with a variance as low as that produced by the Autosampler (See Figure 6). The
MAST data has a higher variance overall, but as protein concentration increases it produces
data of acceptable variance.
DISCUSSION AND CONCLUSIONS
The MAST autosampling system has demonstrated its potential to be a quick and reliable
automatic sample dispensing device, although there are still some issues to be worked out.
Perhaps with more scan data creating a larger sample size more methods in correcting for
droplet variation can be found. Currently with the correction factor applied the resulting
data curves begin to resemble the precision of the autosampler system, but work still needs
to be done to make the resulting data reach the same level of reproducibility as the current
autosampler system.
Some ideas for how this can be realized are a dual needle system, where a second needle
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is mounted beneath the autosampler needle so that the sample when dispensed would be
attracted to the lower needle. Then by moving the upper autosampler needle or by drawing
sample back into the needle, the droplet shape can be controlled. In addition to increasing
the precision of the measurements by reducing the variation in the shape of the droplet,
perhaps this system can be used to alter the shape of the droplet in a way to reduce the
overall curvature of the outside of the drop permitting the beam to better pass through the
sample. This project so far has been a proof of concept as it has demonstrated that the
MAST system is a viable option as an upgrade to the current system, but like all prototypes
there are still a few problems to work out before becoming a complete replacement of the
autosampler.
ACKNOWLEDGMENTS
This work was made possible by funding from the United States Department of Energy
through the Summer Undergraduate Laboratory Internship (SULI) Program. I want to
thank my mentor, Thomas M. Weiss, for giving me the opportunity to work at SLAC, for
his help which permitted the completion of the project, and for exposing me to the projects
being undertaken at the SSRL.
REFERENCES
[1] Jacques, David A., and Jill Trewhella. ”Small-angle Scattering for Structural BiologyExpanding the Frontier While Avoiding the Pitfalls.” Protein Science 19.4 (2010): 642-57.
Print.
[2] Martel, A., Liu, P., Weiss, T. M., Niebuhr, M. & Tsuruta, H. (2012). J. Synchrotron
Rad. 19, 431-434.
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[3] McPhillips, T. M., McPhillips, S. E., Chiu, H.-J., Cohen, A. E., Deacon, A. M., Ellis, P.
J., Garman, E., Gonzalez, A., Sauter, N. K., Phizackerley, R. P., Soltis, S. M. & Kuhn,
P. (2002). J. Synchrotron Rad. 9, 401406. Print
[4] Neylon C (2008) Small angle neutron and X-ray scattering in structural biology: recent
examples from the literature. Eur Biophys J Biophys Lett 37:531541.
[5] Putnam, Christopher D., Michal Hammel, Greg L. Hura, and John A. Tainer. ”X-ray
Solution Scattering (SAXS) Combined with Crystallography and Computation: Defining Accurate Macromolecular Structures, Conformations and Assemblies in Solution.”
Quarterly Reviews of Biophysics 40.03 (2007): n. pag. Print.
[6] Woodward, Roger P. ”Surface Tension Measurements Using the Drop Shape Method.”
First Ten Angstroms (n.d.): 1-6. Web.
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FIGURES
Figure 1: Diagram depicting Synchotron SAXS
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Figure 2: The sample syringe (B) is positioned by a triaxial system of motors (A) which
extracts samples from a 96-sample well tray (C). It dispenses into the capillary tube holder
(D) which is positioned in the path of the X-Ray beam by a length of tubing filled with
a working fluid connected to a flowcell. Solutions such as the sample and wash fluids are
removed from the target area by a tube under the sample stage (E).
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Figure 3: This next generation autosampler operates in a similar manner as the autosampler
in Figure 2 but has several modifications that permit droplet based sample dispensing. The
needle is at a lower position (A) than the original as to permit the formation of a small
droplet directly into the beam. The small aluminum tube at position (B) is an evaporation
guard intended to slow the droplet’s evaporation rate. Also the sample is returned to an
empty capillary position
Figure 4: Image A shows the results of positioning of the light source shining directly at the
microscope objective produces a droplet image with hard edges permitting precise analysis
of the droplet’s size and shape. Image C demonstrates an improper droplet dispensing while
Image B is what the droplet should look like.
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Figure 5: On the left is a plot of two droplet files of 2.5 mg/ml Lysozyme in 2% buffer each
of which is subtracted against 3 buffer files. The graph on the right depicts a corrected data
plot which compensates for differences in droplet size and reduces the percentage variation
between the different curves from 37.87% to 23.95%.
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Figure 6: These are two plots demonstrating a comparison between data collected by the
Autosampler and data collected using the MAST system. Here a concentration series of
Lysozyme from 0.3125mg/ml to 10.00 mg/ml is plotted by a log(I) vs. s (Logarithmic Plot)
and scaled. The MAST data has more variance overall between the different curves even
after scaling than the Autosampler data.
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Figure 7: These plots depict the diminishing noise in the graphed sample data as the concentration of a Lysozyme sample in 2% glycerol increases from 0.3125mg/ml to 10.00 mg/ml.
These graphs are plotted by a log(I) vs. s (Logarithmic Plot) and the samples were taken
on the MAST sampling system.
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TABLES
Sampling Device
Concentration
Average Rg
Standard Deviation
% Error
Autosampler
Autosampler
Autosampler
Autosampler
Autosampler
MAST
MAST
MAST
MAST
MAST
MAST
0.625 mg/ml
1.25 mg/ml
2.50 mg/ml
5.00 mg/ml
10.00 mg/ml
0.3125 mg/ml
0.625 mg/ml
1.25 mg/ml
2.50 mg/ml
5.00 mg/ml
10.00 mg/ml
14.9
14.7
14.5
14.5
14.4
15.6
16.0
15.5
15.0
14.8
14.8
0.15
0.28
0.11
0.07
0.09
0.59
0.56
0.03
0.22
0.03
0.01
0.99
1.89
0.74
0.48
0.63
3.8
3.5
0.2
1.4
0.2
0.04
Table 1: These are the Rg calculations off of lysozyme in 2% glycerol buffer solutions.
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