Simple optimization procedure for objective lens correction collar setting

Abstract

We present a new method for setting a coverglass correction collar on an objective lens. Axial scans across the interface between the specimen volume and the slide are used together with a quantitative function of merit to determine the optimum setting of the correction collar. The method, which simplifies the adjustment for the user and reduces photobleaching, was implemented within the software environment of a scanning microscope system.

Immersion oil for high‐resolution live‐cell imaging at 37°C: optical and physical characteristics

“…First, the optimal setting of the correction collar that compensates for cover slip thickness has to be determined for each new preparation. Part of this problem can be overcome by implementing a semi-automated optimization procedure to set the correction collar (Schwertner et al, 2005), but this is not a standard software feature on most confocal microscopes. Second, evaporation of the immersion water droplet during long time-lapse and/or multiposition experiments poses a serious problem.…”

Light sheet adaptive optics microscope for 3D live imaging

“…As the sample lies generally in a water medium behind the coverslip, this effect is often observed when using a high numerical aperture objective lens with oil immersion. The use of microscope objectives with an adjustable collar, which have been initially designed for correcting the spherical aberration induced by the coverslip thickness variation also offers a way of correcting refractive index mismatch [38].…”

Automated adjustment of aberration correction in scanning confocal microscopy

“…From the observation in the rz image, two correction quality measures are used to evaluate each z image as a function of r, according to [3],…”

“…A manufacturer of confocal microscopes recommends users to maximize the intensity of the interface reflection between the coverslip and specimen to find the best adjustment [2]. Schwertner et al [3] define correction quality measures of these reflections of the clear interface without any specimen, which are L p norms, e.g. image sharpness if p = 2, L 2 [4], and find the optimal adjustment by Gaussian fitting.…”