Autofocus solutions are available for microscopy applications to compensate for spatial drifts induced by mechanical or thermal fluctuations. These solutions are based on laser or LED light reflection at an interface; they all suffer from low contrast and moderate accuracy.
Here we reconsider the fundamental origin of the optical feedback signal, and take crucial advantage of both intensity and phase of the light to achieve unprecedented positioning speed and accuracy along all 3 spatial directions.
KEY BENEFITS vs. STATE OF THE ART
• Both intensity and phase of a reference object linked to the sample are monitored allowing maximum recovery of information far beyond the diffraction limit
• No need to integrate different subsystems to achieve full 3D positioning
• Accuracy better than 10 nm achieved through the analysis of the phase signal, which is enabled by axial focus positioning (key requirement for PALM/STORM ultramicroscopy applications)
• System can easily be integrated into existing optical microscopes; a single lateral port with C-mountis required
• Illumination is set by the conventional optical lamp or LED; there is no need for external laser source. The choice of illumination wavelength is completely defined by the user, therefore avoiding disturbance in the microscope imaging
• Characterized prototype
• Proof of concept on PALM/STORM ultramicroscopy
• Live cell optical microscopy
• Optical inspection