Summary Silicon microchips with thin electron transparent silicon nitride windows provide a sample support that accommodates both light-, and electron microscopy of whole eukaryotic cells in vacuum or liquid, with minimum sample preparation steps. The windows are robust enough that cellular samples can be cultured directly onto them, with no addition of a supporting film, and no need to embed or section the sample, as is typically required in electron microscopy. By combining two microchips, a microfluidic chamber can be constructed for the imaging of samples in liquid in the electron microscope. We provide microchip design specifications, a fabrication outline, instructions on how to prepare them for biological samples, and examples of images obtained using different light-, and electron microscopy modalities. The use of these microchips is particularly advantageous for correlative light-, and electron microscopy.

Correlative fluorescence microscopy and transmission electron microscopy (TEM) is a state-of-the-art microscopy methodology to study cellular function, combining the functionality of light microscopy with the high resolution of electron microscopy. However, this technique involves complex sample preparation procedures due to its need for either thin sections or frozen samples for TEM imaging. Here, we introduce a novel correlative approach capable of imaging whole eukaryotic cells in liquid with fluorescence microscopy and with scanning transmission electron microscopy (STEM); there is no additional sample preparation necessary for the electron microscopy. Quantum dots (QDs) were bound to epidermal growth factor (EGF) receptors of COS7 fibroblast cells. Fixed whole cells in saline water were imaged with fluorescence microscopy and subsequently with STEM. The STEM images were correlated with fluorescence images of the same cellular regions. QDs of dimensions 7 × 12 nm were visible in a 5 μm thick layer of saline water, consistent with calculations. A spatial resolution of 3 nm was achieved on the QDs.

Nanoparticle-based therapy represents a novel and promising approach to treat glioblastoma, the most common and lethal malignant brain cancer. Although similar therapies have achieved significant cytotoxicity in cultured glioblastoma or glioblastoma stem cells (GSCs), the lack of an appropriate approach to monitor interactions between cells and nanoparticle-based therapies impedes their further clinical application in human patients. To address this critical issue, we first obtained NOTCH1 positive GSCs from patient-derived primary cultures. We then developed a new imaging approach to directly observe the dynamic nature of nanoparticles at the molecular level using in situ transmission electron microscopy (TEM). Utilizing these tools we were able to visualize real-time movements of nanoparticles interacting with GSCs for the first time. Overall, we show strong proof-of-concept results that real-time visualization of nanoparticles in single cells can be achieved at the nanoscale using TEM, thereby providing a powerful platform for the development of nanotherapeutics.

We present a novel microfluidic platform to examine biological assemblies at high-resolution. We have engineered a functionalized chamber that serves as a "nanoscale biosphere" to capture and maintain rotavirus double-layered particles (DLPs) in a liquid environment. The chamber can be inserted into the column of a transmission electron microscope while being completely isolated from the vacuum system. This configuration allowed us to determine the structure of biological complexes at nanometer-resolution within a self-contained vessel. Images of DLPs were used to calculate the first 3D view of macromolecules in solution. We refer to this new fluidic visualization technology as in situ molecular microscopy.

Liquid-cell electron microscopy is a rapidly growing field in the imaging domain. While real-time observations are readily available to analyze materials and biological systems, these measurementshave been limited to the two-dimensional (2-D) image plane. Here, we introduce an exciting technical advance to image materials in 3-D while enclosed in liquid. The development of liquid-cell electron tomography permitted us to observe and quantify host− pathogen interactions in solution while contained in the vacuum system of the electron microscope. In doing so, we demonstrate new insights for the rules of engagement involving a unique bacteriophage and its host bacterium. A deeper analysis of the genetic content of the phage pathogens revealed structural features of the infectious units while introducing a new paradigm for host interactions. Overall, we demonstrate a technological opportunity to elevate research efforts for in situ imaging while providing a new level of dimensionality beyond the current state of the field.