Specimen induced aberrations can have detrimental effects in all types of high-resolution microscope. In this study, we present a sensorless technique that uses a deformable mirror (DM) to correct aberrations of both the system and sample. Using a laser-free confocal microscope, with patterned disk illumination and detection. The system is based on a commercial confocal module (Clarity, Aurox Ltd., UK) that uses Light Emitting Diode (LED) illumination to obtain optically sectioned 3D images. The results obtained show that the setup was able to correct aberrations of biological samples used in the study. These systems will help researchers working on various biological systems to obtain improved quality images when focussing deep into thick specimens.
When imaging a sample, inhomogeneities in refractive index cause blur in the image and decrease resolution. Adaptive optics (AO) is a technique that can correct for the resulting aberrations. The most common implementation of AO uses a single deformable mirror that is conjugate to the pupil. A single pupil-conjugate corrective device provides correction over a limited field of view owing to field-dependent aberrations. To overcome this limitation, an additional specimen-conjugate deformable mirror can be used. However, adding a second reflective correction device significantly increases system complexity. We have developed a closed-loop multiconjugate AO system for field-dependent aberration correction in a confocal fluorescence microscope. A 140-actuator deformable mirror is used in the pupil plane with a custom 37-element transmissive deformable phase plate inserted in a sample-conjugate plane. Both devices are calibrated and controlled in closed-loop using a Shack-Hartmann sensor in combination with an integral control law. The sensor consists of an EMCCD and lenslet array with a 500 μm pitch and a 47 mm focal length. Results from a Drosophila ovary and HeLa cells are presented.