Image Scanning Microscopy with STED, Adaptive Optics, and Fluorescence Lifetime Imaging
Overview
This project combines several modern microscopy techniques into a single, versatile scanning microscope. The goal is to push spatial resolution in all three dimensions while preserving the photons emitted by the sample and extending the information recovered from each measurement to include fluorescence lifetime.
Image scanning microscopy and lifetime imaging
Image scanning microscopy (ISM) provides high lateral resolution with minimal photon losses. The sample is scanned by a focused laser beam, and the signal is recorded by an array of detector elements. Reassigning the signal from each element computationally recovers a high resolution image.
The method can be combined with fluorescence lifetime imaging (FLIM). For each detector element, the arrival time of the detected photons is recorded. These arrival times reveal the lifetime of the excited fluorophores and thereby provide a contrast channel sensitive to the local sample composition and molecular environment. Fast single-photon avalanche diode (SPAD) array detectors make this combined spatial and temporal readout possible.
Resolution enhancement with STED
Stimulated emission depletion (STED) allows a further resolution enhancement, limited only by the maximum light intensity the sample can tolerate. The underlying principle is an effective narrowing of the scan focus: a second, high-intensity laser beam depletes the excited fluorophores at the rim of the focal spot, so that only the central region emits. Specific beam configurations enable depletion in the lateral or the axial direction. The lateral resolution gain provided by ISM can therefore be complemented by an axial resolution gain from STED.
Adaptive optical correction
Imaging deeper layers of a sample introduces optical aberrations in both the excitation beam and the detection signal, degrading resolution and signal level. These aberrations can be compensated with adaptive optical elements. In this setup, we integrate such elements and develop efficient algorithms to measure and apply the required adaptive correction.
Outlook
By uniting ISM, STED, adaptive optics, and FLIM in one scanning platform, the setup aims to deliver three-dimensional super-resolution together with quantitative lifetime contrast, even in optically demanding, thicker samples.