Super-resolution PAM by stimulated depletion

Photoacoustic microscopy (PAM) is a microscopy technique relying on the photoacoustic effect. The photoacoustic effect describes thermoelastic generation of acoustic waves by absorption of photons and is employed for structural, functional and molecular imaging. In optical-resolution PAM (OR- PAM) microscale resolution is obtained by using focused laser spot excitation. The resulting photoacoustic signal, i.e. an ultrasonic wave, is measured by using piezoelectric detection. In OR- PAM, the lateral resolution is defined by the diameter of the focal spot and is, therefore, diffraction limited. In axial direction, the resolution is limited by the bandwidth of the ultrasonic detection. In order to improve the axial and lateral resolution in photoacoustic microscopy, various non-linear effects were suggested. Although better resolutions can be achieved by these non-linear techniques, the resolution is still limited by the size of the focus. In the proposed project we intend to break this limitation by using depletion methods. It was shown that in optical fluorescence microscopy subdiffraction resolution in lateral and axial direction can be achieved by using techniques like stimulated emission depletion (STED). In STED microscopy, a diffraction limited laser spot excites chromophores whilst a second laser quenches excited molecules at the periphery of the excitation spot. The fluorescence is thereby confined to the center of the laser spot. The aim of the project is to introduce depletion techniques, such as STED, to photoacoustic microscopy. Thereby, the lateral and axial resolution in photoacoustic microscopy will be brought into the sub-diffraction-region. In particular, the proposed project is devoted to the investigation of the basic principles of photoacoustic depletion microscopy, the search for suitable chromophores, and the demonstration of the feasibility of the method. In the proposed method, the origin of the photoacoustic signal is determined by the location of the depleted focus. As a consequence, no high-bandwidth ultrasonic detection is necessary. We propose a photoacoustic microscopy technique based on sinusoidal intensity modulation of a continuous-wave laser-diode for excitation. The resulting harmonic photoacoustic waves are detected by a narrow-band detection technique. Using a laser-diode as excitation source allows a compact system at relatively low costs, which still facilitates a high signal-to-noise ratio.

No additional funding sources recorded.