Visualization of apoptotic cells using scanning acoustic microscopy and high frequency ultrasound

The goal of this project is to investigate changes in the acoustical properties of cells undergoing cell death for the development of a method for tissue apoptosis detection using high frequency ultrasound (10-60 MHz). A scanning acoustic microscope (SAM) was used for visualization of individual cells undergoing apoptosis (SASAM, Fraunhofer IBMT, Germany). The use of the SAM offers high resolution (1 m spot size) and therefore enables the exploration of acoustical properties of the cell nucleus. Cells were labeled with H33342 and DIOC 3(5) for visualizing condensed chromatin and membranes in fluorescence microscopy. In addition the same cell lines interrogated microscopically were investigated using high frequency ultrasound Recorded radio frequency (rf) data were analyzed using ultrasound spectroscopy. Integrated backscatter coefficients and attenuation values were computed for two cell lines: HeLa and MDCK. Both cell lines responded to the applied chemotherapeutic age nt by apoptosis, assessed by fluorescence microscopy. Acoustical and optical microscopy using the SASAM system clearly enabled a differentiation between apoptotic cells and cells not responding to the treatment. Apoptotic cells displayed a higher contrast in the acoustic images and were less regular in shape. Optical images of the same cells showed nuclear condensation and membrane disruption. Spectral parameters estimated from rf ultrasound showed a 100% increase in the integrated backscatter coefficients for HeLa and MDCK. Attenuation values were increased by 50% to 70% for both cell lines as a function of treatment time. The results of this investigation provide a better understanding of changes in the acoustical properties of cells with cell death and thus to the development of a non-invasive method for measuring the treatment response of tumors using acoustic waves

Gigahertz optoacoustic imaging for cellular imaging

Photoacoustic imaging exploits contrast mechanisms that depend on optical and thermomechanical properties of optical absorbers. The photoacoustic signal bandwidth is dictated by the absorber size and the laser pulse width. In this work we demonstrate that photoacoustic signals can be detected from micron and sub-micron particles. We anticipate applications to include cellular imaging with nanometer sized contrast agents such as gold nanoshells, nanorods, and nanocages. An existing acoustic microscopy system was used (the SASAM 1000, kibero GmbH). This platform is developed on an Olympus IX81 optical microscope with a rotating column that has an optical condenser for transmission optical microscopy and an acoustic module for the acoustic microscopy. The adapted optoacoustic module consists of a Qswitched Nd:YAG solid-state-laser (Teem Photonics, France) generating sub-nanosecond pulses. Scans were acquired of microparticles (1 µm black Toner particles) and cells. The confocal arrangement allowed high signal to noise ratio photoacoustic signals (>30 dB) to be detected at approximately 400 MHz. The particles of various sizes produced signals of different frequency content. In imaging mode, the full width half maximum (FWHM) was measured to be 3.6 µm for the 400 MHz transducer which is in general agreement theory for a 0.3 NA objective (4.3µm). Moreover, images are generated from single melanoma cells, generated by the endogenous contrast from the intracellular melanin