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.

Photoacoustic microscopy is a microscopy technique relying on the photoacoustic effect. The photoacoustic effect describes generation of acoustic waves by absorption of photons and is employed for structural, functional and molecular imaging. In optical-resolution photoacoustic microscopy, microscale resolution is obtained by using focused laser spot excitation. The resulting photoacoustic signal, which is an ultrasonic wave, is measured by a piezoelectric transducer. The lateral resolution is then defined by the diameter of the focal spot and is, therefore, limited by the so-called Abbe limit. It was shown that in optical fluorescence microscopy sub-Abbe resolution can be achieved by using techniques like stimulated emission depletion (STED). In STED microscopy, which development was awarded with the Nobel Prize in 2014, a laser spot excites chromophores whilst a second laser selective deactivates molecules at the periphery of the excitation spot. The fluorescence is thereby confined to the center of the laser spot and thus the resolution is enhanced. The aim of the project was to introduce similar techniques to photoacoustic microscopy in order to enhance the resolution. Within the project, a new superresolution technique SMoQ microscopy (superresolution microscopy using saturated modulation quenching) was developed. In this technique, a focused intensity-modulated laser beam and a donut shaped continuous wave beam are confocally raster-scanned over the sample. Both beams are absorbed by molecules. Sufficiently high intensities of the donut beam lead to saturation of the fluorophores in the outer rim, which enables resolution enhancement. This new imaging method can be applied in fluorescence microscopy and in photoacoustic microscopy. A multimodal microscope, which can measure fluorescence and photoacoustic signals simultaneously, was developed during the project and formed the basis for the SMoQ microscope. The developed multimodal microscope uses a laser-diode as excitation source, thereby allowing a compact system at relatively low costs. Additionally, a new method to assess the quantum yield, i.e. a measure of the efficiency of photon emission, of molecules was proposed.