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A Sealed Preparation for Long-Term Observations of Cultured Cells: Details of Support Slide Construction

Adapted from "A Sealed Preparation for Long-Term Observations of Cultured Cells," Chapter 18, in Live Cell Imaging (eds. Goldman and Spector). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2005.

The aluminum support slide can be fabricated by a professional machine shop. The dimensions of the slide are shown in Figure 1 ; it consists of a 1 x 3-inch aluminum slide 3 mm thick, with a square hole cut in its center and sized to allow a 22 x 22-mm coverslip to slightly overlap the margins of the hole. The ridges going across the ends of the slide on both the top and bottom are necessary to provide clearance for the top and bottom coverslips so that they do not drag on the stage. These ridges are put on both top and bottom surfaces so that a preparation can be moved between an upright and an inverted microscope without having to be rebuilt. However, there is one compromise with this chamber design. When used with upright microscopes for transillumination observations (e.g., phase contrast or differential interference contrast), the cells sit slightly higher above the stage than they would on a conventional glass slide. For upright microscopes, this may make it difficult to achieve perfect Koehler illumination for transmitted light with high-numerical-aperture (NA) condensers, because the condenser travel is often limited by the microscope manufacturer to the level of the stage. To avoid this difficulty, one of two strategies can be used. The first strategy is to use long-working-length condensers with NAs of 0.6 or less. This chamber has most often been used with objectives having magnifications of 40X or less, thus the condenser numerical aperture has not been limiting. In situations in which a condenser of high NA is needed, the setscrew above the condenser focus dovetail can be removed to allow the condenser to come slightly above the stage.

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Figure 1. Dimensions of the aluminum support slide.