KEYWORDS: Adaptive optics, Super resolution microscopy, Microscopes, Super resolution, 3D image processing, Stereoscopy, Optical aberrations, Microscopy, Luminescence, Imaging systems
Super-resolution microscopy allows the observation of sub-cellular structures with a resolution beyond diffraction limit of conventional fluorescence microscopy. However, most super-resolution microscopes have a limited imaging depth due to the inhomogeneous refractive index of the sample that leads to optical aberrations. Adaptive optics has been successfully adopted by many imaging techniques, including 3D Structured illumination microscopy (SIM). We use a fast deformable mirror to modulate the wavefront of fluorescence to compensate for optical aberration and changing focus position at the same time. Adaptive optics successfully extends the depth, the range and the speed of 3D-SIM imaging.
Aberrations are a common problem in microscopes resulting in compromised imaging contrast and resolution. Adaptive optics (AO) can correct aberrations but requires either a wavefront sensor or a wavefront-sensorless AO method that requires multiple sample exposures.
We created a machine learning (ML) approach that embeds physical understanding of the imaging process into a sensorless AO method. This enables correction of aberrations with as few as two sample exposures. The method was translated across different microscope modalities. This includes two-photon microscopy and three-photon microscopy of in vivo mouse neural activity, showing robustness to specimen motion and activity related intensity variations.
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.
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.
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