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: debkelly@vt.edu; 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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