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Contrasting techniques for tissue culture

A wide variety of cell types are cultured in vitro, including primary cells recently obtained from tissue, immortalized cell lines, and stem cells. These different types of cells present similar imaging challenges.

An obstacle to seeing cells is their generally high degree of transparency, which restricts the usefulness of standard brightfield transmitted light microscopy. For this reason, tissue culture microscopes are often equipped for phase contrast imaging – a technique that uses interference to translate subtle differences in optical path length into contrast (even in mostly transparent specimens). Importantly, phase contrast is compatible with plastic culture vessels, unlike polarization-dependent techniques such as differential interference contrast (DIC).

Phase contrast makes identification of single cells relatively easy compared to brightfield, even at low magnification (objective lenses with a magnification between 4 – 20X are most often used for tissue culture). However, phase contrast imaging requires specialized objective lenses and a corresponding annular mask in the illumination light path. Furthermore, there are various types of phase contrast objectives to choose from.

Nikon’s CFI Achromat Series for Apodized Phase Contrast objective lenses are useful for tissue culture observation thanks to the provided reduction in the halo artifact that arises in areas with large optical path differences. This makes it easier to identify the borders of cells and to accurately observe larger model systems, such as tissues and whole organisms, compared to standard phase contrast objectives.

Fluorescence imaging and tissue culture

Fluorescence imaging is becoming a more-and-more routine requirement in the tissue culture laboratory. For example, a common task is checking the expression of fluorescent protein fusions by transfected cells – important for evaluating transfection efficiency, proper localization, and expression levels. Fluorescent stains are also widely used for visualizing key subcellular components (e.g. Hoechst dyes for staining nuclei in live cells).

The ECLIPSE Ts2-FL features up to three different user-selectable LED illumination modules which are built into the microscope. The microscope automatically remembers which LED to use for each filter cube position, and the last used brightness setting. Nikon also offers an optional Contrast Shield accessory for helping to block background ambient light, which is frequently a problem in brightly-lit culture rooms.

The importance of size and comfort

Tissue culture work is often repetitive, resulting in a need for an ergonomically correct system that prioritizes ease of use and economy of motion. The ECLIPSE Ts2 controls are grouped in zones depending on if they are used for transmitted light illumination (left side of the microscope), epifluorescence (right), or both (center).

Tissue culture microscopes are often placed inside biosafety cabinets in order to promote a sterile environment and help protect cell health. Thus, a compact microscope can be advantageous for such work. Additionally, the stage is positioned low on the body, making repeated sample exchange more ergonomic to perform from a sitting position compared to microscopes with stages that are positioned higher up on the body.