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.
KEYWORDS: Microscopy, Luminescence, Spatial resolution, 3D image processing, Confocal microscopy, Two photon excitation microscopy, Stereoscopy, Point spread functions, Objectives, Time lapse microscopy
<p>Two-photon excitation microscopy is one of the key techniques used to observe three-dimensional (3-D) structures in biological samples. We utilized a visible-wavelength laser beam for two-photon excitation in a multifocus confocal scanning system to improve the spatial resolution and image contrast in 3-D live-cell imaging. Experimental and numerical analyses revealed that the axial resolution has improved for a wide range of pinhole sizes used for confocal detection. We observed the 3-D movements of the Golgi bodies in living HeLa cells with an imaging speed of 2 s per volume. We also confirmed that the time-lapse observation up to 8 min did not cause significant cell damage in two-photon excitation experiments using wavelengths in the visible light range. These results demonstrate that multifocus, two-photon excitation microscopy with the use of a visible wavelength can constitute a simple technique for 3-D visualization of living cells with high spatial resolution and image contrast.</p>