This dilemma sent the ibidi founders on a search for a material that would have the optical properties of glass but, at the same time, would allow for good cell adherence and growth. They eventually discovered a polymer foil with excellent optical quality that became the ibidi Polymer Coverslip. It could be bonded to an upper structure, and this combination was the first ibidi microscopy chamber. This special polymer foil, however, was very hydrophobic and the cells would not adhere to it. Luckily, the founders discovered that the polymer foil could be physically treated to fix the problem, and the special surface treatmentibiTreat was born!

The thickness of the coverslip plays an important role in the imaging quality, because most microscope objective lenses are designed to focus on a specimen on top of a coverslip with a certain thickness (Fig. 1). In cell microscopy, the standard coverslip thickness is 0.17 mm (170 µm +20/–10 µm), which corresponds to a #1.5 coverslip. While most polystyrene tissue culture plates and dishes have a bottom thickness of 500–1000 µm, the ibidi Polymer Coverslip has the same thickness as a standard #1.5 glass coverslip.

Refractive Index

The refractive index (which measures the speed of light inside a specific material) of an ibidi Polymer Coverslip is 1.52, and therefore the same as the refractive index of glass. In contrast, standard tissue culture plates and dishes made from polystyrene have a refractive index of 1.58. This value is important because microscopy objectives are designed for coverslips with a refractive index of 1.52.

Fig. 1: Microscopic lenses are optimized for the standard coverslip thickness of 170 µm (left). Using other coverslip thicknesses makes it impossible for high resolution objective lenses to focus on the specimen (right).

To provide high-quality imaging results, the transmission ability of microscopy carriers has to be high across a variety of different wavelengths. This is the case for glass as well as for the ibidi Polymer Coverslip. Standard polystyrene materials block the UV light, which makes them not really suitable for all microscopy techniques and fluorophores (Fig. 2).

Fig. 2: Transmission curves of various coverslip and cell culture materials across different wavelengths.

The autofluorescence properties of the microscopy carrier material can disturb the imaging process by showing up as a high background signal during microscopy. While the autofluorescence of the ibidi Polymer Coverslip material and glass is very low, cultureware made from polystyrene has a very high autofluorescence (Fig. 3).

Fig. 3: Autofluorescence intensity for different coverslip and cell culture materials at different wavelengths.

First of all, plastic is softer than glass, which makes its surface elasticity more similar to in vivo tissue (Discher et al., 2005;
Fig. 4).

Tissue-Culture Treatment

Secondly, in contrast to glass, the surface of plastic can be modified in a way that makes it more hydrophilic, which most adherent cell types love. This physical surface treatment (which is not a coating) is commonly known as tissue culture treatment. Most plastic vessels used for cell culture are modified in this way. At ibidi, we call the tissue culture surface modification of our Polymer Coverslip, "ibiTreat," which is not a protein coating, but a physical surface treatment that enhances the attachment of the cells. Of course, additional protein coatings can be applied to the uncoated ibidi Polymer Coverslip or the ibiTreat version without compromising the optical quality.

Fig. 4: Surface elasticity of different tissues as well as coverslip and cell culture materials.