The development of affinity capture devices—a nanoscale purificationplatform for biological in situ transmission electron microscopy
Katherine Degen,a Madeline Dukes,b Justin R. Tannerc and Deborah F. Kelly*c
Received 22nd November 2011, Accepted 12th December 2011
DOI: 10.1039/c2ra01163h
The use of in situ transmission electron microscopy (TEM) to study biological processes is a new
frontier in high-resolution imaging. Here we report the development of affinity capture devices that
are used to isolate biological entities for in situ studies. Affinity capture devices are silicon nitride
microchips coated with functionalized lipid monolayers. We tested the isolation capacity of the
devices by using protein synthesis machinery as a test specimen for single particle and in situ TEM
analysis. Overall, we demonstrate the feasibility of purifying biological assemblies in a liquid
environment within a TEM column. This novel application may serve to bridge the gap between
cellular and molecular imaging techniques.
Introduction
The purification of biological complexes remains the rate-
limiting step for structural studies in the electron microscopy
(EM) field.1 One key issue is that macromolecular assemblies
need to be rapidly removed from other cellular contaminants
while maintaining their native functionality. Classical biochem-
istry techniques rely upon lengthy chromatographic or gradient
separations and require large amounts of starting material.
Often, weakly associated subunits of multi-component assem-
blies readily dissociate during purification.
To address these issues, we have recently developed the affinity
grid approach. Affinity grids are carbon coated EM grids
containing functionalized monolayers doped with Ni–NTA
(nickel–nitrilotriacetic acid) lipids (Avanti Polar Lipids,
Alabaster, Alabama).2 Affinity grids mimic a Nickel–NTA
affinity column to purify proteins carrying a continuous stretch
of histidine residues (His tag).3 His-tagged proteins bind with
high affinity to Ni–NTA matrices.4 Moreover, affinity grids
differ from conventional column separations in that they require
only nanogram quantities of starting material and they can
purify protein complexes in less than 5 min.2
New materials, such as silicon nitride5 and graphene,6 have
been introduced as alternatives to traditional carbon substrates
to coat metal EM grids. Recently developed liquid-flow sample
holders used for in situ TEM utilize silicon nitride membranes as
windows for their flow-cell chamber.7 Therefore, we aimed to
adapt our nanoscale protein purification system to work with
silicon nitride surfaces.
Here we present a novel expansion of our affinity capture
technology. We demonstrate the use of functionalized semicon-
ductor devices to purify active protein machinery for structural
studies and in situ imaging. This new application for silicon
nitride membranes gives rise to EM affinity capture devices.
These devices can be used as the flow-cell windows for
commercial in situ TEM sample holders. Utilizing this platform
we were able to capture tagged macromolecules of interest while
viewing them in a liquid-flow environment within a TEM
column. Additionally, our new application maintains a rapid
purification component with high specificity and nanogram
sensitivity. The method also lends itself for use with antibodies
against cell surface proteins to isolate whole cells. The isolation
of rare cells or cancer cells may be possible in combination with
TEM imaging. This opens a new avenue for the visual screening
of therapeutic interventions aimed at multiscale imaging—from
the molecular to the cellular levels. Overall, the use of affinity
capture devices and live TEM imaging provide a unique platform
to view active biological processes at nanometer resolution.
Results and discussion
The functionalization of silicon nitride devices
Affinity capture devices were developed using custom designed
silicon nitride microchips having dimensions of 2.60 mm 6 2.00 mm
(Fig. 1a). The devices contain a central 50 nm thick window that is
500 mm 6 50 mm and allows the electron beam to penetrate the chip
for imaging purposes (Fig. 1b).7 These microchips are commercially
available from Protochips, Inc., (Raleigh, NC) at www.protochips.
com/products/durasin.html.
Lipid mixtures composed of 2% to 20% Ni–NTA lipids and
DLPC (1,2-dilauryl-phosphatidylcholine) filler lipids (Avanti
Polar Lipids, Alabaster, Alabama) were each reconstituted in
chloroform (1 mg ml21) and cast over 25 ml drops of Milli-Q
aDepartment of Biomedical Engineering, University of Virginia,Charlottesville, VA, U. S. A.bApplications Science, Protochips, Inc., Raleigh, NC, U. S. A.cVirginia Tech Carilion Research Institute, 2 Riverside Circle, Roanoke,VA, U. S. A. E-mail: [email protected]; Fax: 01 540 985 3373;Tel: 01 540 526 2031
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water placed into wells of Teflon blocks as previously described.8
The Teflon blocks were sealed in a humid petri dish and
incubated on ice for 60 min. Silicon nitride chips were heated to
150 uC for 90 min and allowed to cool to room temperature prior
to incubating on top of the lipid layers for at least 1 min. The
hydrophobic lipid chains interacted favourably with the silicon
nitride surface and the lipid layer was effectively transferred to
the chip (Fig. 1c). The chips and associated lipid layers were
lifted off of the drop and used to isolate His-tagged protein
targets (Fig. 1d).
The purification of macromolecules using affinity capture devices
We used His-tagged ribosomes produced in E. coli as an ideal EM
specimen to test the purification capacity of our affinity capture
devices. Bacterial cell lysates containing recombinant His-tagged
subunits (rpl3) incorporated into 50S ribosomes were prepared in
the presence of imidazole. Briefly, a His-tagged construct for a
subunit of the large ribosome, rpl3 (clone MPMGp800 I10582)
was purchased from RZPD (GmbH, Berlin, Germany) with the
T7 promoter in the PQE30NST vector. His-tagged rpl3 was
expressed in E. coli (BL21 strain) overnight at 16 uC in 200 ml of
LB media containing 30 mg l21 kanamycin and 50 mg l21
ampicillin upon the addition of 0.5 mM IPTG at an OD600 = 0.6.
Cells containing recombinant rpl3 were harvested and
centrifuged at 3500 g for 20 min at 4 uC. Cell pellets were
suspended in 20 ml of lysis buffer containing 20 mM Hepes (pH,
7.5), 150 mM NaCl, and 0.2 g of lysozyme (EMD Biosciences,
Inc., San Diego, CA), followed by sonication. The cell lysate was
centrifuged at 17 000 g for 20 min at 4 uC. Total protein
concentration of the lysate was determined to be 3.2 mg ml21 by
absorbance measurements performed at 280 nm.
To test if our devices could capture macromolecules from
bacterial lysates, we employed a similar approach to that of the
affinity grid system. We applied 4 ml aliquots of bacterial lysate
in buffer solution containing 20 mM Hepes (pH, 7.5), 150 mM
NaCl, 20 mM CaCl2, 20 mM MgCl2 and 60 mM imidazole to
affinity capture devices and incubated the solution for 2 min at
room temperature. Following incubation, excess amounts of
lysate were blotted away using Whatman 1 filter paper and the
chips were washed with successive drops of Milli-Q water then
negatively stained using 1% uranyl formate (Electron Microscopy
Sciences, Hatfield, PA).9
To test the requirement for the presence of Ni–NTA lipids in
the monolayer, samples were prepared on devices using only the
DLPC filler lipids. Also, to test the isolation component of the
system, silicon nitride devices lacking the lipid layer were glow-
discharged and incubated with the 4 ml aliquots of the same
bacterial lysate, then washed and negatively stained using the
same procedures. The glow-discharged devices should reveal the
total contents of the bacterial lysate. We can, therefore, assess
the purification power of the functionalized affinity devices
by directly comparing their contents with respect to the glow-
discharged samples.
EM imaging of purified complexes in a static environment
Negatively stained affinity device specimens were examined using
a FEI Spirit BioTwin TEM (FEI, Hillsboro, OR) equipped with
a tungsten filament and operated at an acceleration voltage of
120 kV under low-dose conditions. Images were recorded on a
FEI Eagle 2k HS CCD camera with a pixel size of 30 mm at
a nominal magnification of 68 0006 and a defocus value of
21.5 mm for a sampling of 4.4 A pixel21.
We determined the optimum working concentration of the
lysate solution to be between 0.2 to 0.01 mg ml21. Lysate samples
containing a total protein concentration above 0.2 mg ml21
recruited an excess of ribosomal complexes onto our affinity
capture devices and were not useful for image processing routines.
Samples prepared with a total protein concentration below
0.01 mg ml21 did not recruit enough particles per image for
reasonable processing routines. On average, we estimated y10 ng
of ribosomes were recruited per device using 4 ml aliquots of lysate
solution (0.2 mg ml21). Thus, our system exhibits nanogram-level
sensitivity with an estimated detection limit of y2.5 ng ml21 per
device. The incorporation of imidazole in our buffer helped to
attenuate non-specific background components from binding to
the surface of our affinity capture devices.
Images of bacterial lysate samples prepared on affinity devices
lacking Ni–NTA lipids did not show any complex binding
(Fig. 2a). This supports the idea that the Ni–NTA lipids are
required in the lipid layer to bind to His-tagged complexes in our
sample. In contrast, lysate samples prepared on glow-discharged
silicon nitride microchips lacking the lipid monolayer contained
an excess of proteins that randomly represent the total cell
contents of the bacteria (Fig. 2b). A minimal level of ribosomal
complexes can be identified in the lysate sample (white circles,
Fig. 2b). Images of lysate samples prepared on affinity capture
devices decorated with 2% Ni–NTA lipid monolayers showed
Fig. 1 Affinity capture devices produced on commercial silicon nitride
microchips (a) contain viewing windows (b) that allow an EM beam to
penetrate to the specimen level. Devices can be functionalized with lipid
monolayers (c) containing Ni–NTA head groups (red circles) that bind to
His-tagged protein targets (red complexes) (d).
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protein complexes with features and dimensions (25 – 30 nm)
consistent with that of bacterial 50S ribosomes (white circles,
Fig. 2c). Considering that aliquots from the same bacterial lysates
were added to each device and we see a more concentrated,
homogeneous population of complexes when the Ni–NTA
monolayer coating is present (compare Fig. 2c with Fig. 2b)
supports our claim that we are specifically purifying tagged
complexes onto affinity capture devices.
We collected 300 images of samples prepared on affinity
capture devices and selected 5919 individual particles from the
images using the WEB interface for the SPIDER software
package.10 Particles were windowed into individual image panels
of 120 x 120 pixels. Using SPIDER, the selected particles were
subjected to 10 cycles of multi-reference alignment. References
used for the first round of alignment were randomly selected
from the raw images. Each round of alignment was followed by
principal component analysis and K-means classification, out-
putting class averages.10
Representative averages (Fig. 2d, top panel) were filtered to
20 A resolution and cross-correlated with projections of the 50S
ribosome crystal structure (pdb code, 1ML511) also filtered to
the same resolution (Fig. 2d, middle panel) using the SPIDER
software package. Normalized cross-correlational coefficients
above 0.7 indicate an adequate match between the experimental
averages and the theoretical projections.2 A comparison between
the experimental averages (Fig. 2d, top panel) and the theoretical
projections (Fig. 2d, middle panel) gave mathematical correla-
tion values above 0.8. This calculation statistically quantifies the
measure of structural similarities exhibited by the 50S complexes
isolated on our devices with respect to their known correlates. In
general, single particle EM samples have a nanogram detection
limit, which is a highly sensitive means to determine the identity
of bound complexes. As an additional note of validation, we
compared corresponding views of the filtered 3D volume of the
50S crystal structure11 (Fig. 2d, bottom panel) with the
experimental projections (Fig. 2d, top panel) and found a high
degree of agreement. Taken together, these results support the
finding that the affinity capture system can be successfully used
with silicon nitride devices to prepare specimens suitable for
TEM structural analysis.
In situ TEM imaging of captured biological specimens
The use of in situ TEM to study biological processes is a new
frontier in the imaging field. Until recently, there was no way to
maintain biological specimens in a liquid environment while
enclosing them in the high vacuum system of a TEM. The newly
developed TEM liquid-flow holder7 accommodates biological
samples between two semiconductor microchips that are tightly
sealed together. These chips form a microfluidic device at the tip of
a specimen holder that permits liquid flow in and out of the holder.
To test whether affinity capture devices could be used to
isolate biological macromolecules within a TEM column, affinity
devices were prepared using 20% Ni–NTA lipid layers and
allowed to dry overnight in a grid box at room temperature.
Fig. 2 Images of negatively stained bacterial lysate samples applied
affinity devices lacking Ni–NTA lipids in the monolayer (a) show no
specific binding. (b) Glow-discharged, non-functionalized silicon nitride
devices show an excess of randomly bound proteins while only a minimal
number of complexes (white circles) can be identified. Scale bar is 50 nm.
(c) Image of cell lysate applied to functionalized affinity capture devices
show protein complexes consistent with the size and features of 50S
ribosomes (white circles). (d) Representative averages of affinity captured
specimens indicate the presence of 50S ribosomes (top panel). Class
averages show a high correlation (. 0.8) with filtered projections of the
50S ribosome crystal structure (pdb code, 1ML5;11 middle panel). A
comparison between the representative averages (top panel) and the
filtered 3D volume and crystal structure of the 50S ribosome (red,
bottom panel) indicate a high agreement in different orientations.
Individual panels are 52 nm. Scale bar is 15 nm.
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Based on our previous results in preparing cryo-EM specimen,
we anticipated a 10-fold increase in the available number of
Ni–NTA functional head groups may be necessary to ensure
complex binding. One functionalized affinity device and one
non-functionalized device having a spacer size of 150 nm were
loaded into the tip of a Poseiden flow-holder (Protochips, Inc.,
Raleigh, NC) designed for a JEOL 1400 TEM. The TEM,
located at the Georgia Institute of Technology (Atlanta,
Georgia), was equipped with a tungsten filament operating at
120 kV and a Gatan Orius SC1000 CCD camera. Images were
recorded at a nominal magnification of 50 0006 to 100 0006using a defocus value of approximately 23 mm.
A 500 ml aliquot of the same lysate sample that was used for
single particle analysis was loaded into the specimen flow-holder
using a step-motor pumping system that regulated the solution
flow rate to be 300 ml h21. We acquired images of the affinity
capture devices before the lysate solution entered the imaging
window of the holder as well as an initial time point before
complex binding occurred (Fig. 3a). No notable complexes were
present at the initial time point of solution flow. As the lysate
solution containing tagged complexes flowed over the affinity
device, we observed an optimal level of decoration (Fig. 3b)
within 5 min that became saturated shortly thereafter (Fig. 3c)
due to the excess of Ni–NTA lipid used in comparison to the
negatively stained specimens. This suggests that dynamic capture
events could potentially be modulated at the level of Ni–NTA
incorporation during the functionalization step. A magnified
view of the affinity device (100 0006) shows complexes with the
dimensions consistent with 50S ribosomes (Fig. 3d).
In general as we imaged many different areas within the
transparent viewing window of the affinity devices, we noted a
consistent binding pattern as complexes in the lysate solution
flowed across the device and accumulated. The characteristic
nature of the dynamic binding events typically included: 1)
minimal to no particles binding; 2) optimal binding; 3) saturation
of bound complexes and 4) oversaturation. Differing combina-
tions of these steps can be visualized within a single imaged area
(Fig. 4). After particle binding was completed, buffer solution
could flow into the holder with no appreciable reduction in the
number of complexes bound to the functionalized affinity device.
We speculate that bound complexes could be further diluted in
the flow holder by increasing the concentration of imidazole in
the flowing sample buffer. Overall, complex binding events could
be influenced by the initial number of tagged macromolecules
present in the lysate, by the solution flow rate or by the amount
of Ni–NTA lipids used to coat the silicon nitride devices. These
observations are consistent with our previous work in the
development of affinity grids.2 Therein, we noted that an increase
in the amount of Ni–NTA lipids used in the production of EM
affinity grids corresponded with an increase in the number of
particles bound to the functionalized grid.
To test the potential use of affinity capture devices to recruit
cells for in situ imaging experiments, we used HEK 293T cells
and a series of adaptor proteins. His-tagged Protein A and
monoclonal antibodies (IgGs) against the Notch receptor were
purchased from Abcam (Cambridge, MA). HEK 293T cells were
grown to confluency in Dulbecco’s modified Eagle’s medium and
mechanically dislodged from the culture dish with the tip of
a pipette. Affinity capture devices containing 2% Ni–NTA
monolayers were prepared and coated with 0.05 mg ml21 of His-
tagged Protein A for 1 min at room temperature. Subsequently,
antibodies (0.1 mg ml21 in 20 mM Hepes (pH, 7.5), 150 mM
NaCl, 20 mM CaCl2, 20 mM MgCl2 and 60 mM imidazole) were
added to the devices coated with Protein A and incubated for
1 min. Cells contained in conditioned media were added to the
devices and incubated on the decorated devices for 2 min.
Specimens were then washed and negatively stained. A schematic
Fig. 4 A dynamic population of tagged 50S ribosomal complexes were
captured upon affinity devices. Images across many regions showed a
disparate number of 50S particles bound to the functionalized devices.
Fig. 3 Tagged ribosomes were captured from a bacterial lysate solution
and imaged in an in situ flow holder within a TEM column. (a) Upon the
initial flow of lysate solution into the imaging window, no notable
ribosomal complexes adhered to the capture device (50 k6). (b) Tagged
complexes bound to the functionalized affinity capture devices as they
flowed inside the holder until the field of view became saturated with
particles (c). Scale bar is 200 nm. (d) Higher magnified view (100 k6) of
the complexes bound to the affinity device showed dimensions consistent
with 50S ribosome complexes. Scale bar is 100 nm.
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representation of an affinity capture device with tethered cells is
given in Fig. 5a.
As a negative control, Ni–NTA containing affinity devices
were prepared and coated with His-tagged Protein A but not IgG
antibodies. 293T cells were added to the devices and incubated
for up to 5 min at room temperature. Using TEM, no cells were
identified on any of the devices (Fig. 5b). This finding supports
the necessity for the use of antibodies to bind to receptors on the
cells’ surfaces. Upon the addition of IgG to protein A coated
microchips, cells were recruited to the affinity capture devices
and could be imaged using TEM (Fig. 5c). Images taken at
approximately 90006 magnification allowed us to confirm the
presence of whole cells on our devices. At higher magnification
(50 0006) we could begin to discern the presence of internal
complexities (Fig. 5d). In order to fully explore any underlying
cellular architectures, cells with a thickness of , 2 microns
should be used in conjunction with a TEM operating at higher
voltages. Overall, we anticipate affinity capture devices would be
able to recruit cells in an in situ environment, similar to our
purification experiments performed on 50S ribosomes. We are
currently focusing our work in this direction.
Conclusions
Here we present the necessary steps for producing functionalized
silicon nitride surfaces for EM analysis. These functionalized
microchips, that we have named affinity capture devices, can be
used to isolate protein complexes for single particle EM
structural studies. Samples may be examined in a static
environment by embedding the biological material in heavy
metal salts, such as uranyl formate, or in a dynamic liquid
environment using a TEM flow-holder.
With the recent success of in situ TEM experiments5, the
potential exists for the use of affinity capture devices to view
biological processes in a liquid environment. One limitation in
viewing reactions, however, may be attributed to electron beam
damage. Low-dose imaging conditions help to minimize this
damage while examining biological specimens. In addition, the
slow but steady flow of liquid within the microfluidic specimen
chamber may also help to reduce damage to beam-sensitive
samples. Overall, we anticipate that affinity capture devices can
be used to observe dynamic interactions with in situ imaging just
as affinity grids can be used to prepare single particle specimens
for cryo-EM. Furthermore, our work shows the first images of
macromolecular complexes being isolated in a liquid environ-
ment while fully enclosed in an EM column.
Multiscale applications for affinity capture approaches
At the molecular level, we demonstrate the use of affinity capture
devices to isolate biological assemblies for single particle
imaging. This approach may soon allow us to view molecular
events in real-time. At the intermediate scale of view, receptors
or antibodies may be added to affinity capture devices while
viruses or other infectious agents are flowed, captured and
imaged. At the cellular level, antibodies against cell surface
markers can be bound specifically to affinity devices and used to
isolate rare cells types including stem cells and cancer cells. It
may also be possible to perform single cell experiments with
broad scientific applications on a single device. Therefore, the
use of affinity capture devices may permit biologists to observe
real-time events from the structural level to the cellular scale in a
near-native, liquid environment. Overall, these new molecular
tools will undoubtedly enable us to view biological processes in a
remarkable new way.
Acknowledgements
We would like to acknowledge Stephen Mick for his advice and
support of our work.
References
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Fig. 5 (a) Schematic to indicate how cells (yellow) may be recruited to
functionalized affinity capture devices containing His-tagged protein A
and antibodies (IgGs, blue) against cell surface receptors. (b) Affinity
devices lacking IgGs fail to recruit cells. (c) Image of HEK 293T cell
isolated onto an affinity device. Scale bar is 0.5 mm. (d) Image taken on
the curved edge of the cell surface shows visible features within the cell.
Scale bar is 0.1 mm.
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