In-vivo observation of cells with a combined high-resolution multiphoton-acoustic scanning microscope

We present a combined multiphoton-acoustic microscope giving collocated access to the local morphological as well as mechanical properties of living cells. Both methods relay on intrinsic contrast mechanisms and dispense with the need of staining. In the acoustic part of the microscope, a gigahertz ultrasound wave is generated by an acoustic lens and the reflected sound energy is detected by the identical lens in a confocal setup. The achieved lateral resolution is in the range of 1 mum. Contrast in the images arises mainly from the local absorption of sound in the cells related to viscose damping. Additionally, acoustic microscopy can access the sound speed as well as the acoustic impedance of the cell membrane and the cell shape, as it is an intrinsic volume scanning technique. The multiphoton image formation bases on the detection of autofluorescence due to endogenous fluorophores. The nonlinearity of two-photon absorption provides submicron lateral and axial resolution without the need of confocal optical detection. In addition, in the near-IR cell damages are drastically reduced in comparison with direct excitation in the visible or UV. The presented setup was aligned with a dedicated procedure to ensure identical image areas. Combined multiphoton/acoustic images of living myoblast cells are discussed focusing on the reliability of the method.