Optical Properties of High Resolution Microscopy

No. 1.5 Coverslip-Like Thickness

The thickness of the coverslip is a crucial aspect of imaging quality. The typical thickness of a coverslip is 0.17 mm (170 µm, No. 1.5). Most of objective lenses used for microscopy are corrected to this special thickness. Thinner or thicker substrates require the use of correction collars on the objective lenses, which then prevents the formation of blurred images by spherical and chromatic aberrations.

The special plastic material (ibidi Standard Bottom) that is used in all ibidi μ-Slides, μ-Dishes and μ-Plates provides coverslip-like optics for high resolution microscopy. The μ-Dishes^{35 mm, high} are also available with a coverslip glass bottom.

Compatibility to Immersion Oil

Oil immersion is an important technique that is used to increase the resolution of the objective, up to its physical limit. Placing immersion oil instead of air between the objective lens and the coverslip allows a significantly higher amount of light to be collected by the lens. This, in turn, increases the resolution and the signal-to-noise ratio in microscopic images.

Cell culture chambers for high resolution microscopy need to be compatible with immersion oil. All ibidi μ-Slides, μ-Dishes and μ-Plates fulfill this important requirement.

Numerical Aperture

The numerical aperture (NA) of an objective lens summarizes the range of angles over which a lens can collect light. NA is one of the main parameters of an objective lens, which defines resolution and luminous intensity.

The numerical aperture is defined by NA = n sin θ, where n is the refractive index of the medium in which the lens works (e.g., 1.52 for immersion oil) and θ is the half-angle of the maximum cone of light that can enter into the lens.

Transmission

Transmission is a crucial parameter for microscopy. It describes a material’s ability to permit the passage of light through it at specific wavelengths. The more light that is absorbed, the less it can contribute to fluorescence excitation and image acquisition. Unlike normal cell cultureware, which is not transparent in UV light region below 300 nm, the ibidi Standard Bottom also permits light transmission in wavelengths below 300 nm.

Low Fluorescence Back­ground (Autofluorescence)

Autofluorescence is a material property that describes the intrinsic fluorescence value of the pure material. The fluorescence signal comes from the material and contributes to the imaging process as noise. This can be annoying when trying to image faint fluorescence signals. Depending on the wavelength and type of material, all materials show some degree of autofluorescence signals.