Using a combination of silicone and urethane resin, we established a rapid technique for preparing living specimens for microscopy. One major advantage of this technique is that the coverslip is rigidly attached and does not detach during handling. As a result, it is possible to continuously observe living cells at high magnification and resolution using an oil immersion objective. Another advantage is that living cells are quickly confined to the space between the glass slide and coverslip, protecting them from environmental changes, which can cause serious effects on cell function and morphology. Moreover, high-resolution observations of real-time responses of cells are possible, using the combination of the mounting technique and a simple flow chamber.

Several kinds of equipment have been developed in order to observe the fine structure and real-time responses of living cells under the microscope. One technique is to grow cells in culture dishes for viewing with an upright microscope with a water dipping objective. The water dipping objective has the advantage of allowing direct observation of cells on any type of culture dish, but these objectives may not be readily available in many laboratories and the resolution is likely to be lower than that possible with an oil immersion objective. Alternatively, glass-bottom dishes and chambers are widely used for observing living cells, mainly with oil immersion objectives on inverted microscopes. This equipment makes it possible to observe living cells at high resolution without further sample preparation and has the advantage that the dishes can be returned to the incubator for future analysis. However, glass-bottom plasticware needs relatively large volumes of reagents to treat cells and can be expensive for frequent use.

A less-expensive method is to use a glass slide and coverslip; as a result, techniques for mounting coverslips are widely employed in short-term studies of living cells (1). In the conventional method, a coverslip is inverted onto a glass slide and all sides of the coverslip are sealed using paraffin, VALAP (a 1:1:1 mixture of petroleum jelly, lanolin, and paraffin), or nail polish in order to prevent cells from drying out (2,3,4). However, when the mounting substance does not completely adhere, the coverslip can sometimes slide, especially when using an oil immersion objective. For all of the methods currently in use, the attachment of the coverslip to the glass slide is not secure enough to permit convenient, reliable observation using high-resolution objectives.

To find an alternative to traditional mounting methods, we directed our attention to caulking compounds that are compatible with biological materials. We found that a combination of silicone and urethane resin worked as a quick and rigid adhesive without causing any visible damage to cells. Only plastic adhesive tape, silicone, and urethane resin are required for this method, meaning any lab can easily adopt the technique. It is not necessary to use medical-grade materials: silicone products intended for coating glassware and urethane resin sold at craft stores are both adequate. The steps for coverslip mounting are described in our protocol (see protocol at www.BioTechniques.com) and illustrated in Figure 1. Briefly, the edges of a square glass coverslip are pretreated with silicone so that a 5-mm coated boundary around the edge of the glass is formed. Cells are grown on the coverslip, which is then inverted onto a reservoir formed on a microscope slide by tape–urethane resin walls and the glass bottom of the slide. In initial tests, we found that coverslips secured in this way were strong enough to support a suspended 20-g weight for >30 min without leakage of enclosed liquid (data not shown). Importantly, adhesion of the coverslip to the slide was strong and lasting enough so that it is possible to use mounted coverslips with oil immersion objectives on both upright and inverted microscopes without slippage.

Figure 1. Diagram of specimen preparation. (Click to enlarge)

Maximizing observation times of living cells requires not just a securely mounted coverslip. Rapid specimen preparation is also critical for maintaining healthy conditions for cells. It can take 5–6 min to prepare specimens by the traditional methods (e.g., using nail polish). During sample preparation, temperature changes can occur and cause damage to the cells. Even when a temperature-controlled stage is used for observation, this delay sometimes causes cell shrinkage and it may take >1 h for recovery (Figure 2A). A long recovery time precludes experiments in which cells are pulsed with a stimulus and then mounted for microscopy, given that several biologically important responses occur within a few minutes of stimulation.

Figure 2. Comparing the effect of the mounting techniques on living cells. (Click to enlarge)

On the other hand, using the silicon-urethane resin mounting technique, the procedure is completed in <1 min and cells are not seriously damaged. Almost all cells are healthy (Figure 2B), so it is possible to start observation within a few minutes. As shown in Figure 2C, it was possible to keep the cells active for 30 min.

The coverslip method is convenient for shorter-term observation of cells, but longer-term experiments require control of appropriate temperature, oxygen, and pH. Additionally, it is desirable in many experiments to be able to add a reagent and wash out excess while continuously observing cell responses. There are several simple flow chambers designed to maintain optimal cell conditions by continuously exchanging a medium at a low flow rate, and that allow reagents to be added into the chamber (4,5) (Japanese patent serial no. 2006-69789). Some of these chambers can be handmade from basic materials (e.g., glass slide and acrylicresin). The silicone-urethane technique can be adapted to these flow chambers, and the combination makes it possible to observe the real-time responses of cells at high resolution using an oil immersion objective. Figure 3A shows a simple flow chamber that was made in-house using the silicone-urethane coverslip mounting approach. In a model experiment, internalization of a fluorescent ligand was examined under a fluorescence microscope (Figure 3B). When a fluorescent ligand is used, it is necessary to remove the excess ligand in order to examine cell response. By increasing the flow rate in the flow chamber, the ligand was washed out within 15 s, so early cellular responses were able to be observed. It was possible to observe the cells for 3 h, and during that time no leakage of the medium enclosed between the chamber and the coverslip occurred. If the coverslip is handled very carefully with tweezers, it is also possible to peel the coverslip from the chamber. [alternatively, silicone grease (TORAY, Tokyo, Japan) can be used instead of urethane resin to attach the coverslip, although the adhesive strength is somewhat weaker]. Once the coverslip is removed, it can be returned to a culture dish for additional cultivation for further observations at a later time point.

Figure 3. Time lapse imaging of living cells using a simple flow chamber made with the mounting technique. (Click to enlarge)

In summary, we describe a simple and inexpensive technique to easily and securely mount specimens for live-cell observation using oil immersion objectives. The technique can also be used with fixed samples, in which case we have found that it is possible to keep a specimen viable for >1 year for repeated observations.