Letter pubs.acs.org/JPCL
Nanofluidic Cells with Controlled Pathlength and Liquid Flow for Rapid, High-Resolution In Situ Imaging with Electrons C. Mueller,† M. Harb,‡,§ J. R. Dwyer,‡,∥ and R. J. Dwayne Miller*,†,⊥ †
Departments of Chemistry and Physics, University of Toronto, 80 St. George Street, Toronto, ON M5S 3H6, Canada Insight Nanofluidics, Inc., 60 St. George Street Suite 331, Toronto, ON M5S 1A7, Canada § MAX-lab, Lund University, P.O. Box 118, Lund, Sweden ∥ Department of Chemistry, University of Rhode Island, 51 Lower College Road, Kingston, Rhode Island 02881, United States ⊥ Max Planck Research Group for Structural Dynamics, Department of Physics, and Centre for Ultrafast Imaging, University of Hamburg, c/o DESY, Notkestrasse 85, 22607 Hamburg, Germany ‡
S Supporting Information *
ABSTRACT: The use of electron probes for in situ imaging of solution phase systems has been a long held objective, largely driven by the prospect of atomic resolution of molecular structural dynamics relevant to chemistry and biology. Here, we present a nanofluidic sample cell with active feedback to maintain stable flow conditions for pathlengths varying from 45 nm to several 100 nm, over a useable viewing area of 50 × 50 μm. Using this concept, we demonstrate nanometer resolution for imaging weakly scattering polymer and highly scattering nanoparticles side by side with a conventional transmission microscope. The ability to flow liquids allows control over sample content and on-the-fly sample exchange, opening up the field of high-throughput electron microscopy. The nanofluidic cell design is distinguished by straightforward, reliable, operation with external liquid specimen control for imaging in (scanning) transmission mode and holds great promise for reciprocal space imaging in femtosecond electron diffraction studies of solution phase reaction dynamics. SECTION: Liquids; Chemical and Dynamical Processes in Solution
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environments are usually circumvented by adopting sample preparation techniques based on sectioning or cryoprotection. While these widely accepted techniques are important in enabling imaging, there is always a question of deformation of the object of interest in the freezing process and mechanical action of microtoming. Ideally one would like to observe structures in their native liquid environment with minimal perturbation. Moreover, aside from questions of the effect of freezing on the sample structure, static and frozen samples severely limit the ability to observe structural dynamics. In situ imaging of systems in liquid environments enables covering the full range of dynamics and correlated motions of great interest in problems from protein folding to nucleation growth and selfassembly. The importance of high-resolution transmission electron microscopy (TEM) imaging in the liquid-phase is reflected in the variety of approaches presented to date delivering solution phase capabilities.16−22 However, in situ sample exchange and liquid flow for high throughput imaging with high spatial resolution has not been achieved to date. Most approaches for TEM imaging in liquid are either based on the use of an aperture-restricted, differentially pumped
ne of the long-standing goals in chemistry is to attain insight into reaction dynamics on an atomic level. This objective requires femtosecond time resolution and subangstrom spatial resolution to follow barrier crossing dynamics.1 With the recent developments of high-brightness femtosecond electron1−3 and X-ray sources,4,5 both resolution limits have become experimentally accessible. In the case of electron structural probes, the scattering cross section for typical electron energies is so high that samples must be prepared with path-lengths on the 100 nm scale.6 For solid state samples, a number of ingenious methods have been developed, mainly by the electron microscopy community, for preparing samples in that thickness regime.6 The importance of attaining similar nanoscale pathlengths for liquid environments has long been recognized and is of particular importance in electron microscopy7−9 and other techniques that use electrons to deliver unparalleled high-resolution imaging, for applications in chemistry, biology, and materials and medical sciences.10−12 Recent developments in this field have allowed for the discovery of key nanoscale structural features of molecular assemblies such as protein−DNA binding complexes,13 of biomolecule−nanoparticle aggregates,14 and of cells and their contents.15 Yet, high-resolution real-time imaging of structures and processes in liquid environments remains challenging, especially if active sample exchange through flow is desired. We note here that limitations to studying samples in liquid © 2013 American Chemical Society
Received: May 23, 2013 Accepted: June 26, 2013 Published: June 26, 2013 2339
dx.doi.org/10.1021/jz401067k | J. Phys. Chem. Lett. 2013, 4, 2339−2347
The Journal of Physical Chemistry Letters
Letter
Figure 1. Depictions of major approaches of liquid cells for TEM applications. (A) static cell with spacer material (orange), (B) flow cell with the liquid layer thickness defined through application of polymer suspension droplets in the corners of the substrates (orange), and (C) nanofluidic sample cell as presented in this work containing a defined flow path, inlet and outlet ports implemented into the cell, and a liquid layer thickness defined by the rigid spacer material (orange).
Figure 2. Experimental setup. Schematic of the feedback loop to control the liquid flow conditions within the nanocell. Notice that the fluidic feedthroughs connected to the TEM column deliver sample to the nanocell while the TEM is under vacuum and in operation.
sample volume,17 or on the availability of so-called electrontransparent windows to define a physical sample cell.19−21 Different schemes for achieving nanoscale liquid layers are shown in Figure 1. Hermetic sealing of small chambers (Figure 1A) outside the electron microscope is a common approach, but inherently requires time-consuming sample chamber
exchange as soon as the enclosed sample volume should be exchanged to examine a new sample or the same sample under different conditions.9,19−21 Recently, Grogan et al. used two ultrathin silicon nitride windows within their “nanoaquarium” approach,20,23 and Alivisatos et al. fabricated an even thinner enclosure between graphene layers to achieve high-resolution 2340
dx.doi.org/10.1021/jz401067k | J. Phys. Chem. Lett. 2013, 4, 2339−2347
The Journal of Physical Chemistry Letters
Letter
Figure 3. Micrographs from the HD-2000 STEM (200 keV) taken with dark field imaging mode using 45 nm spaced nanocells: (A) single gold nanorods in liquid suspension inside the nanocell; (B) 5/10 nm gold nanoparticles, 8 × 8 × (30−60) nm gold nanorods and 36/210 nm polymer based nanoparticles drop-cast on the external surface of the imaging window of a 45 nm spaced nanocell; (C/D) line profiles parallel and perpendicular to the gold nanorod axis marked in A/B. The scale bars in A and B were enhanced for better readability.
flow channel within the nanofluidic cell allows for the desired ease of use and exchange of samples in situ, in both static and flowing liquid environments. We tested the performance of our nanofluidic cell using conventional TEMs operating with different electron beam settings and in different imaging modes. High-resolution imagingon a nanometer scalecan be preserved, even for low atomic number samples (e.g., biological and polymer-based samples), but requires ultrathin, ∼100 nm liquid layer thicknesses. These specifications are also essential for solution phase femtosecond electron diffraction that will enable atomic level views of solution phase chemistry. The capability for rapid, yet well-controlled sample exchange, in contrast to poorly defined multiple flow paths depicted in Figure 1B, is essential to ensure identical conditions for pump−probe protocols for this class of experiments. In the present studies, liquid was introduced into the cell simply by connecting a syringe to the holder’s inlet tubing external to the vacuum column. Upon entering the imaging area, the liquid front could be clearly seen by the degradation of image resolution and by reduced electron transmission. The key to establishing the designed cell path-length was to stabilize the flow during cell filling, using negative feedback with a signal related to the liquid layer thickness within the cell viewing volume. This principle was first demonstrated within twodimensional infrared spectroscopy studies on liquid water using
imaging.21 In comparison to these fully enclosed chambers, de Jonge et al. overcame sample isolation with a fluidic sample cell with micrometer-thick liquid layers allowing free spreading of the liquid underneath the imaging windows (Figure 1B).24,25 However, the performance of such a sample cell is limited by the reproducibility and stability of the liquid layer thickness between the two silicon nitride viewports: a sample thicker than ∼100 nm requires either an increase in electron energy or energy filtering, with a concomitant loss of signal, to combat the resolution losses. The nanofluidic cell introduced here overcomes the severe technical challenges described above and allows for routine application with high throughput for sample introduction and exchange, and high-resolution imaging. The cell depicted in Figure 1C delivers samples with a tunable, yet well-defined and reproducible liquid film thickness. This is achieved by designing a rigid and chemically resistant spacer to permit accurate path length control, directional flow, and stability in both static and pressure-driven fluid conditions.26 The fluid channel in our cell allows for controlled, rapid, on-the-fly exchange of
