In situ TEM of Biological Assemblies in Liquid

Summary

Here we describe a procedure to image viral complexes in liquid at nanometer resolution using a transmission electron microscope.

Abstract

Researchers regularly use Transmission Electron Microscopes (TEMs) to examine biological entities and to assess new materials. Here, we describe an additional application for these instruments- viewing viral assemblies in a liquid environment. This exciting and novel method of visualizing biological structures utilizes a recently developed microfluidic-based specimen holder. Our video article demonstrates how to assemble and use a microfluidic holder to image liquid specimens within a TEM. In particular, we use simian rotavirus double-layered particles (DLPs) as our model system. We also describe steps to coat the surface of the liquid chamber with affinity biofilms that tether DLPs to the viewing window. This permits us to image assemblies in a manner that is suitable for 3D structure determination. Thus, we present a first glimpse of subviral particles in a native liquid environment.

Introduction

A common goal of biologists and engineers is to understand the inner-workings of molecular machines. Transmission Electron Microscopes (TEMs) are ideal instruments to visualize these intricate details at near-atomic resolution^1-2. In order to sustain the high vacuum system of a TEM, biological samples are typically embedded in thin films of vitreous ice³, sugars⁴, heavy metal salts⁵, or some combination thereof⁶. As a result, images of embedded specimens may reveal only limited snapshots of dynamic processes.

Early attempts to maintain biological specimens hydrated in environmental liqu....

Protocol

1. Prepare Affinity Capture Devices¹⁶

1. Clean the silicon nitride E-chips by incubating them in 15 ml of acetone for 2 min followed by 15 ml of methanol for 2 min (Figure 1A). Allow chips to dry under laminar air-flow.
2. Incubate the dried chips on a heated stir plate (without stirring) for 1.5 hr at 150 °C, then allow them to cool to room temperature before use.
3. Use Hamilton syringes to compose lipid mixtures in small glass tubes to contain 25% chloroform, 55% DLPC (1,2-dilauroyl-phosphocholine) in chloroform (1 mg/ml) and 20% Ni-NTA lipid (1,2-dioleoyl-....

Results

Representative images of DLPs in liquid using E-chips that were glow-discharged (Figure 3A) show fewer DLPs in a given viewing area, presumably due to diffusion, in comparison to DLPs that are enriched on Affinity Capture devices (Figure 3B). The addition of uranyl formate in the imaging chamber enhances the contrast of the specimen and hence the visibility of individual DLPs in solution (Figure 3C, top panel). Better contrast allows for downstream image processing such .......

Discussion

In our presented work, we employed the affinity capture approach to tether rotavirus DLPs to a microfluidic platform. This allowed for in situ imaging of macromolecular complexes in a liquid microenvironment. The capture approach is significant with respect to other microfluidic imaging techniques because it localizes biological specimens to the imaging window to negate large diffusive issues that arise while recording images in liquid. However, one of the most critical steps in our protoc.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
E-chips, spacer chip	Protochips, Inc.	EPB-52TBD	400 μm x 50 μm window
E-chip, top chip	Protochips, Inc.	EPT-45W	400 μm x 50 μm window
Ni-NTA lipid	Avanti Polar Lipids	790404P	Powder form
DLPC (12:0) lipid	Avanti Polar Lipids	850335P	Powder form
Volumetric flasks	Fisher Scientific	20-812A; 20-812C	1 ml; 5 ml
Hamilton Syringes	Hamilton Co.	80300, 80400	1-10 μl; 1-25 μl
Whatman #1 filter paper	Whatman	1001 090	100 pieces, 90 mm
Glass Petri dishes	Corning	70165-101	100 mm x 15 mm
Glass Pasteur pipettes	VWR	14673-010	14673-010
Glass culture tubes	VWR	47729-566	6 mm x 50 mm
Acetone	Fisher Scientific	A11-1	1 L
Methanol	Fisher Scientific	A412-1	1 L
Chloroform	Electron Microcopy Sciences	12550	100 ml
His-tagged Protein A	Abcam, Inc.	ab52953	10 mg
Milli-Q water system	EMD Millipore Corp.	Z00QSV001	Ultrapure Water
HEPES	Fisher Scientific	BP310-500	500 g
Equipment
Poseidon In situ specimen holder	Protochips, Inc.		FEI compatible
FEI Spirit BioTwin TEM	FEI Co.		120 kV
Eagle 2k HS CCD camera	FEI Co.		10 Å/pixel sampling at 30,000X
Gatan 655 Dry pump station	Gatan, Inc.		Pump holder tip to 10-6 range
PELCO easiGlow, glow discharge unit	Ted Pella, Inc.		Negative polarity mode
Isotemp heated stir plate	Fisher Scientific		Heat to 150 ºC for 1.5 hr