Microinjection based 3-dimensional imaging of subcellular structures with digital holographic microscopy

摘要

In cell biology one of the most important but also most difficult tasks is to visualize dynamic 3-dimensional intracellular processes and alterations. In combination with an ordinary microscope, digital holography provides contact-less, marker-free, quantitative phase contrast imaging to record both long term time-lapse investigations in toxicology and cancer research and fast dynamic processes like shape variations. However, the rather homogenous cellular refraction index limits the technology in case of the imaging of intracellular structures and processes.rnHere we demonstrate the first results of the selective alteration of the cytoplasmatic refraction index in order to enhance the intracellular contrast. Therefore we combined a commercial inverted microscope with the digital holography setup and a microinjection unit. The microinjection unit was used to inject a small amount of glycerol (50 % in water) to increase the intracellular refraction index. The injection process, the flow, the spreading and the final mixing of glycerol with the surrounding medium could be visualized by digital holography. Non diffractive reconstruction algorithms enable digital holographic focus adjustment with constant imaging scale, multi focus imaging of the object planes and subsequent focus correction. We injected adherent A549 tumor cells in a perinuclear region with the glycerol-water mixture. The reconstructed images indicate an impermeability of the nuclear membrane for glycerol resulting in an enhanced intracellular contrast. The specific alteration of the intracellular refraction index allows the development of a 3-dimensional imaging for dynamic intracellular processes in living cells.