(Developmental Center-Driven Research Project)

UCSF: P Kner, D Agard, J Sedat;

Weizmann Inst. of Science: Z Kam;

LLNL: S Olivier; UCSC: B Sullivan, J Kubby, J Zhang, O Azucena, J Cao, J Crest, D Gavel

Adaptive optics has had a tremendous impact in astronomy, correcting for aberrations caused by the earth’s atmosphere. This technology is applicable to light microscopy, where sample-induced aberrations degrade the performance of high-resolution imaging in thick specimens. However, the actual implementation requires substantial developmental efforts. The goals of our project are to incorporate adaptive optical elements in a wide-field microscope to solve a number of problems: 1) To correct aberrations due to refractive index mismatch between the immersion medium and the environment of live biological samples; 2) To use the adaptive element to focus through thick samples without moving the sample or the objective, so that three-dimensional focal sectioning microscopy can be carried out at high speeds without moving the sample; 3) To correct for aberrations arising from three-dimensional refractive index variations within the sample; and 4) To develop specialized adaptive elements tailored to microscopy.

These goals need adaptive optical elements with large strokes (0.1mm) and high precision (1/20th λ). To meet these requirements, we have chosen to use the Mirao52D deformable mirror from Imagine-Optic. This mirror can set the focus mode with a stroke greater than 50um.

We have begun by addressing problems 1 and 2. Fig. 1 illustrates the severity of the refractive index mismatch between live samples and an oil immersion objective. Imaging 20µm into a sample with the refractive index of water, the point-spread function is severely degraded. We have designed a system to correct spherical aberrations in high resolution three-dimensional wide-field fluorescence microscopy. Our results demonstrate that this system can focus through and correct spherical aberrations in 30 micrometer-deep samples.