• Funding: FWF (P 27839-N36)
• Volume: EUR 352k
• Duration: 36 months
• Principal investigator: Thomas Berer
• Research Partner: Prof. Thomas A. Klar, Institute of Applied Physics, JKU Linz
• Co-investigators: Gregor Langer, Bianca Buchegger (Institute of Applied Physics, JKU Linz)
• Start: June, 1^st 2015

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 intensity modulation of a continuous-wave laser-diode for excitation. The resulting harmonic photoacoustic waves are detected by a narrow-band detection technique.

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. 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.

In a first step, multimodal optical-resolution photoacoustic and fluorescence microscope in frequency domain was demonstrated. Photoacoustic waves and modulated fluorescence are generated in chromophores by using a modulated diode laser. The photoacoustic waves, recorded with a hydrophone, and the fluorescence signals, acquired with an avalanche photodiode, are simultaneously measured using a lock-in technique (Fig. 1).

With this setup, we demonstrated, e.g., multimodal imaging of a blood smear (see Fig. 2). In the photoacoustic image, the individual red blood cells could be identified on the basis of their donut-like form. In the luminescence image, three bright spots originating from the RBCs centers are visible. These three bright spots correspond to structures which are also visible in the bright-field microscopy image. However from the bright-field microscopy image, one cannot conclude that these three spots have a chemical composition different from the RBCs. By simultaneous photoacoustic and fluorescence imaging we were able to distinguish between these chemically different structures in the blood smear. Blood components of a healthy human do normally not exhibit fluorescence. In case of certain hemoglobinopathies erythrocyte inclusions, like Heinz bodies, basophilic stipplings, or Howell-Jolly bodies, can be found. It is known that at least one of these inclusions, the so called Heinz bodies exhibit luminescence. Heinz bodies occur e.g. in case of thalassemia but also as effect of chronic liver disease, after splenectomy, or due to mineral deficiency. We therefore believe that the presented microscope is well suited for investigations in this field of hematology.

As every substance does absorb light at some wavelength, in principle every substance can be viewed with photoacoustic imaging. However, in practice one is limited by the available excitation wavelengths, which in our case is 405 nm. Therefore, only substances that absorb at 405 nm can be visualized, e.g. hemoglobin. In order to acquire images of cells that are normally transparent to 405 nm light, staining is necessary. In Fig. 3 multimodal imaging of stained biological epithelial cells is shown. As the photoacoustic and the luminescence signal originate from the dye, both images (Fig 3a and b) look similar. This is confirmed by the overlay in Fig 3c.