Notes on Imaging Chambers and Coverslips

When you prepare samples to bring to the Nikon Imaging Center, please ensure that your coverslips (coverglasses) and glass-bottom chambers are # 1.5 thickness. This glass is 170 µm thick (high quality glass will have a tolerance of ± 5 µm) and most microscope objectives are designed for this glass thickness. In fact the coverslip is the first considered lens in an objective design. The thickness of the coverglass is extremely important for using high NA oil-immersion objectives. Multiple companies supply high-tolerance # 1.5 coverslips and imaging chambers. Coverglass thickness # 0 and # 1 should only be used when extra working distance is absolutely essential (they are 100 µm and 150 µm thick, respectively). Notify us to place an objective onto the imaging system to compensate for the non-standard coverglass thickness. It may be necessary to modify the glass chemistry to enhance cell attachment and/or morphology. Poly-lysine, fibronectin, and collagen are all suitable for this purpose. You can apply a simple homogenous layer by pipetting or create a pattern with the PRIMO module on Ray to fully control the substrate shape and biochemisty.

35 mm dishes (e.g. Ibidi, Mattek, and World Precision Instruments), chambered coverslips (e.g. Ibidi and Nunc), multi well plates (e.g. 96-well plate with high tolerance coverglass Cellvis, or 96-well and 384-well plates of Nunc) are compatible with our incubation systems, preferably with a glass bottom with # 1.5 thickness. Note, Ibidi also offers a polymer bottom, but this polymer bottom is not compatible with our standard Nikon immersion oil. Notify us when you use such a polymer bottom, we have Ibidi’s immersion oil that is compatible with the polymer bottom. However, if you wish to perform differential interference contrast (DIC), consider a glass bottom and a glass lid. DIC requires polarized light and the birefringence of most plastics distorts light polarization.

When mounting coverslips on slides, you will get better results by placing the coverslip directly in the middle of the slide. With our inverted microscopes the coverslip faces down and when the coverslip is too far to the end of the slide, your sample will rest on the slide on one side and on the coverslip on the other side. This results in an angle of your sample and affects the quality of tiling experiments. This is especially important for the sample holders of Lana and Sterling. Putting coverslips in the middle will also prevent you from smearing oil onto the sample holders.