Imaging Specific Protein Labels on Eukaryotic Cells in Liquid with Scanning Transmission Electron Microscopy
Diana B. Peckys,1 Madeline J. Dukes,2 Elisabeth A. Ring,1 David W. Piston,1 and
Niels de Jonge1 * 1 Molecular Physiology and Biophysics, Vanderbilt University Medical Center, 2215 Garland Ave, Nashville, TN 37232-0615 2 Department of Chemistry, Vanderbilt University, Nashville, TN 37235; present address: Protochips, Inc., Raleigh, NC 27606
*
niels.de.jonge@
vanderbilt.edu
Introduction Understanding the structure and dynamics of the
protein complexes that underlie cellular function is a central scientific challenge [1]. Biochemical techniques used to identify such complexes would be enhanced by the imaging of specific molecular positions in the context of intact cells, with protein-scale resolution (on the order of a few nanometers). Currently, though, nanometer resolution can only be achieved at the cost of less-direct imaging of the unperturbed cell. Cellular ultrastructure is traditionally studied by transmission electron microscopy
(TEM), which yields
nanometer resolution on embedded and stained sections, or cryo sections [2–4]. Tese cellular samples are neither intact nor in their native liquid state. Light microscopy is used to image protein distributions in fluorescently labeled cells in liquid to investigate cellular function [5], but even recent improvements in resolution by nanoscopy techniques [6, 7] are still insufficient to resolve the individual constituents of protein complexes. Tus, development of techniques capable of high-resolution imaging in native cellular states would contribute significantly to our understanding of cellular function at the molecular level. Te development of liquid compartments that include electron-transparent silicon nitride membrane windows [8] has led to the introduction of a novel concept to achieve nanometer resolution on tagged proteins in cells [9]. Eukaryotic cells in liquid are placed in a microfluidic
chamber, with a thickness of up to 10 µm, contained between two ultra-thin electron-transparent windows, as in Figure 1. Te specimen is then imaged with the scanning transmission electron microscope (STEM). Due to the atomic number (Z) contrast of the STEM, nanoparticles of a high-Z material, such as gold, can be detected within the background signal produced by a low-Z liquid, such as water. Just as proteins tagged with fluorescent labels can be used to study protein distributions in cells with fluorescence microscopy, nanoparticles that are specifically attached to proteins [10] can be used to study protein distributions in whole cells in liquid, but with a much higher resolution. Liquid STEM can also be used in combination with bimodal probes that are visible with both fluorescence and electron microscopy, such as dye-conjugated gold nanoparticles or semi-conductor nanocrystals known as quantum dots (QDs) [11, 12]. An additional advantage of electron microscopy imaging of whole cells in liquid is the compatibility with sample preparation techniques used for
16
Figure 1: The principle of liquid scanning transmission electron microscopy (STEM). Cells kept in liquid are enclosed between two electron-transparent silicon nitride windows. Scanning a focused electron beam over the sample leads to the detection of elastically scattered electrons with an annular dark field detector. Labels of high atomic number materials can thus be distinguished. From [9].
light microscopy, that is, the absence of sectioning, freezing, and staining.
Instrumentation Te main component of the liquid STEM system is a
microfluidic chamber comprised of two silicon microchips, each supporting a 50 nm thick silicon nitride (SiN) window (Protochips, Inc., NC) [13, 14]. Figure 2A shows a scanning electron microscopy (SEM) image of the backside of a microchip. Te dimensions of the microchip were 2.00 × 2.60 × 0.30 mm, and those of the SiN window were 50 × 200 µm. Two microchips were placed in the tip of a specimen holder for liquid flow (Protochips Inc., NC). Te sides of the microchips were manufactured with a precision of ±10 µm with respect to the SiN window position to allow precise alignment of the windows when the two microchips were stacked. Precise alignment of the windows in the microfluidic chamber was needed for the electron beam to be transmitted through both windows, and this was achieved via the use of precision-made alignment poles in the slot of the tip (see Figure 2B). One of the microchips contained a spacer [14], typically 6 µm thick,
doi:10.1017/S1551929511000903
www.microscopy-today.com • 2011 September
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60 |
Page 61 |
Page 62 |
Page 63 |
Page 64 |
Page 65 |
Page 66 |
Page 67 |
Page 68 |
Page 69 |
Page 70 |
Page 71 |
Page 72 |
Page 73 |
Page 74 |
Page 75 |
Page 76 |
Page 77 |
Page 78 |
Page 79 |
Page 80 |
Page 81 |
Page 82 |
Page 83 |
Page 84