Wet electron microscopy (EM) is a new imaging method with the potential to allow higher spatial resolution of samples. In contrast to most EM methods, it requires little time to perform and does not require complicated equipment or difficult steps. We used this method on a common murine macrophage cell line, IC-21, in combination with various stains and preparations, to collect high resolution images of the actin cytoskeleton. Most importantly, we demonstrated the use of quantum dots in conjunction with this technique to perform light/electron correlation microscopy. We found that wet EM is a useful tool that fits into a niche between the simplicity of light microscopy and the high spatial resolution of EM.

Introduction

Microscopy has played a vital role in biology since the days of Robert Hooke, leading to the origin of cell theory (1). First, light microscopy and later, electron microscopy (EM), allowed biologists to explore the cell down to the molecular level. Individual components of the cell, from organelles down to single proteins were imaged using these techniques.

The central issues of microscopy are contrast and resolution. Contrast provides the ability to differentiate structures in the cell from one another and from the background. Resolution refers to the minimum separation between objects required to identify them as individual structures. In order to effectively use microscopy to explore the biological landscape, the sample must have sufficient contrast and resolution.

For light microscopy, the natural contrast of the sample may easily be enhanced through the application of chromophores or fluorophores. This also allows multicolor imaging using differential staining. However, the resolution of light microscopy is physically restricted by the Abbe limit. The Abbe limit dictates a minimum x-y spatial resolution given by λ/2NA, where λ is the light wavelength, and NA is the numerical aperture of the objective lens (2).

By comparison, EM has extremely fine spatial resolution. Using transmission electron microscopy (TEM), distances on the order of 10⁻¹⁰ m or 1 Å may be resolved, and with scanning electron microscopy (SEM), 10⁻⁸ m or 10 nm is possible (3). However, obtaining contrast in EM is more difficult than the simple staining needed for light microscopy. Complicated preparation techniques, including critical point drying, coating, embedding, and sectioning are required (4). Expensive equipment and extensive training are necessary, creating a barrier that prevent many researchers from using EM.

Correlative light/electron microscopy is the ideal technique for structural work, combining nondestructive, time-lapse light microscopy with high spatial resolution EM (5). Unfortunately, the destructive nature of EM sample preparation, combined with the arduous task of finding the same cell previously imaged—makes this an extremely difficult technique in practice.

A new technology available from Quantomix™ allows for biological EM without laborious sample preparation. Their product consists of a scalable capsule with an electron-transparent membrane. The sealed, fully hydrated sample is then imaged through the membrane, which behaves as a coverslip does in light microscopy. Electron-dense stains may be used to enhance sample contrast, as fluorescent stains are used in light microscopy. This technique, dubbed Wet SEM™, makes EM as easy to use as light microscopy, while retaining a substantial resolution advantage (6).

In addition to its uses for biological SEM, this platform is uniquely suited to correlative microscopy. As the membrane is both photon and electron transparent, it is possible to image the same cells in both light and electron microscopy. To provide contrast in both modes of imaging, an electron dense fluorescent stain was needed. Quantum dots fill that role, being made of a CdSe/ZnS fluorescent material (7,8).

Materials and Methods

Cell Culture

The IC-21 cell line (accession no. TIB-186; ATCC, Manassas, VA, USA) used in these experiments is an adherent murine macrophage line. These cells were maintained at 37°C and 5% CO₂ in RPMI media (Mediatech, Herndon, VA, USA), supplemented with 15% fetal bovine serum (FBS; Mediatech) and 50 IU penicillin/50 µg/mL streptomycin (Mediatech). Cells were passaged by detaching in phosphate-buffered saline (PBS) without Ca²⁺ (Mediatech) with a dilution factor from 1:5 to 1:2.5.

IC-21 cells were detached from a culture dish when at approximately 75% confluency. The resulting cells were centrifuged at 600× g for 5 min. The supernatant was aspirated, and the cells resuspended at 2×10⁵ cells/mL concentration. Fifteen microliters of this cell solution were added to a series of QX-102 capsules (Quantomix, Nes-Ziona, Israel). These capsules were secured in a MP-10 multiwell plate (Quantomix). The multiwell plate's reservoirs were filled with sterilized water, and the plate was incubated overnight at 37°C and 5% CO₂.

Cell Staining

After the overnight incubation, the capsules were removed from the incubator. The samples were washed with 1×PBS and fixed in a solution of 4% formaldehyde/0.1% Triton® X-100 in 1×PBS for 15 min. The samples were then washed again with PBS and stained with a contrast-enhancing agent.