Aberrations are known to severely compromise image quality in optical microscopy, especially when high numerical aperture (NA) lenses are used in confocal fluorescence microscopy (CFM) and two-photon microscopy (TPM). The method of adaptive optics may correct aberrations and restore diffraction limited operation. So far the problem of aberrations that occur in the imaging of biological specimens has not been quantified. However, this information is essential for the design of adaptive optics systems. We have therefore built an interferometer incorporating high NA objective lenses to measure the aberrations introduced by biological specimens. The measured wavefronts were decomposed into their Zernike mode content in order both to classify and quantify the aberrations. We calculated the potential benefit of correcting different numbers of Zernike modes using different NAs in an adaptive CFM by comparing the signal levels before and after correction. The results indicate that adaptive correction of low order Zernike modes can provide significant benefit for many specimens. The results also show that quantitative fluorescence microscopy may be strongly affected by specimen induced aberrations in non-adaptive systems.

SummaryConfocal or multiphoton microscopes, which deliver optical sections and three-dimensional (3D) images of thick specimens, are widely used in biology. These techniques, however, are sensitive to aberrations that may originate from the refractive index structure of the specimen itself. The aberrations cause reduced signal intensity and the 3D resolution of the instrument is compromised. It has been suggested to correct for aberrations in confocal microscopes using adaptive optics. In order to define the design specifications for such adaptive optics systems, one has to know the amount of aberrations present for typical applications such as with biological samples. We have built a phase stepping interferometer microscope that directly measures the aberration of the wavefront. The modal content of the wavefront is extracted by employing Zernike mode decomposition. Results for typical biological specimens are presented. It was found for all samples investigated that higher order Zernike modes give only a small contribution to the overall aberration. Therefore, these higher order modes can be neglected in future adaptive optics sensing and correction schemes implemented into confocal or multiphoton microscopes, leading to more efficient designs.

SummarySpecimen-induced aberrations affect the imaging properties in optical 3D microscopy, especially when high numerical aperture lenses are used. Studies on aberrations are often properly concerned with the degradation of image quality such as compromised resolution or reduced signal intensity. Apart from these, aberration effects can also introduce geometric image distortions. The effects, discussed here are particularly strong when thick biological specimens are investigated. Using a high numerical aperture interferometer, we measured wavefront aberrations in transmission mode and quantified geometric distortions associated with specimeninduced aberrations. This assessment for a range of biological specimens allows estimation of the accuracy of spatial measurements. The results show that high-resolution spatial measurements can be significantly compromised by specimeninduced aberrations.

The recording of data in multiple layers, rather than a single layer, permits a significant increase in the capacity of optical data storage devices. However, focusing to the different layers introduces different amounts of depth-dependent aberrations. Variable aberration correction is therefore necessary to maintain diffraction-limited operation. We demonstrate the use of adaptive optics to predict and correct these aberrations for both the recording and read-out of such media.

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.