Liquid Scanning Transmission Electron Microscopy: Imaging Protein Complexes in their Native Environment in Whole Eukaryotic Cells

Abstract

Scanning transmission electron microscopy (STEM) of specimens in liquid, so-called Liquid STEM, is capable of imaging the individual subunits of macromolecular complexes in whole eukaryotic cells in liquid. This paper discusses this new microscopy modality within the context of state-of-the-art microscopy of cells. The principle of operation and equations for the resolution are described. The obtained images are different from those acquired with standard transmission electron microscopy showing the cellular ultrastructure. Instead, contrast is obtained on specific labels. Images can be recorded in two ways, either via STEM at 200 keV electron beam energy using a microfluidic chamber enclosing the cells, or via environmental scanning electron microscopy at 30 keV of cells in a wet environment. The first series of experiments involved the epidermal growth factor receptor labeled with gold nanoparticles. The labels were imaged in whole fixed cells with nanometer resolution. Since the cells can be kept alive in the microfluidic chamber, it is also feasible to detect the labels in unfixed, live cells. The rapid sample preparation and imaging allows studies of multiple whole cells.

Correlative Electron and Fluorescence Microscopy of Magnetotactic Bacteria in Liquid: Toward In Vivo Imaging

“…Cumulative electron doses on the order of magnitude used here (,0.1 electrons?Å 22 ) coincide with a recent fluid cell STEM study of gold nanoparticle uptake into eukaryotic cells; these doses were shown to induce only small changes in the cellular structure with a single STEM exposure 43 . The authors proposed that the first STEM image obtained at the outer cellular region contained information of the ultrastructure of the live cell and subsequent images in other areas likely contained information about the live cell as well.…”

“…Z-contrast imaging. De Jonge and co-workers have established correlative fluid cell STEM and fluorescence microscopy as a powerful technique for studying various aspects of eukaryotic cells, such as locating epidermal growth factor receptors in COS7 cells 38 , exploring the correlation between cellular function and ultrastructure in yeast cells 39 , and investigating electron beam damage and cell viability in live COS7 cells 43 . Other electron microscopy techniques exist for imaging whole hydrated cells, such as environmental scanning electron microscopy (ESEM) 44 , and air SEM (ASEM) 45 .…”

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy

“…Note that the electron dense QD core appears somewhat smaller and rounder than the cores of QDs emitting at 655 nm, consistent with their smaller core size 22 of ~5 nm. a length scale of a few nanometers so that its stoichiometry can be studied within the context of individual, intact cells [4][5][6][7] . This is not possible with (super resolution) light microscopy 23 because its resolution is insufficient to distinguish molecules engaged in a protein complex.…”

“…A new approach was introduced recently, to image whole cells with labeled proteins in liquid state using STEM [1][2][3] , or correlative fluorescence microscopy and STEM [4][5][6][7] . This methodology is capable of studying the spatial distribution of membrane proteins and protein complexes including their stoichiometry at the single molecule level in the intact plasma membranes of whole cells.…”

Opportunities and challenges in liquid cell electron microscopy

“…If the enclosure has suitable size and surface condition, and if nutrients are supplied, unfixed cultured cells can be kept alive in a liquid cell chamber at room temperature with liquid flow for several hours (97,103). But is it possible for cells to remain alive while images are recorded?…”