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pH sensing in living cells represents one of the most prominent topics in biochemistry and physiology. In this study we performed one-photon and two-photon time-domain fluorescence lifetime imaging with a laser-scanning microscope using the time-correlated single-photon counting technique for imaging intracellular pH levels. The suitability of different commercial fluorescence dyes for lifetime-based pH sensing is discussed on the basis of in vitro as well of in situ measurements. Although the tested dyes are suitable for intensity-based ratiometric measurements, for lifetime-based techniques in the time-domain so far only BCECF seems to meet the requirements of reliable intracellular pH recordings in living cells.

Density changes of GeO 2 and SiO 2 glasses subjected to irradiation by tightly focused femtosecond pulses are observed by Raman scattering. It is shown that densification caused by the void formation in GeO 2 glass is very similar to the changes under hydrostatic pressure. In contrast, the experimental observations in SiO 2 glass could be explained by pressure effect or by the fictive temperature anomaly, i. e., a resultant smaller specific volume of the glass (a denser phase) at a high thermal quenching rate. Density changes of GeO 2 and SiO 2 glasses are opposite upon close-to-equilibrium heating; this gives new insights into the mechanisms of densification under highly non-equilibrium conditions: fs-laser induced micro-explosions, heating and void formation. The pressure and temperature effects of glass modification by ultra-short laser pulses are discussed considering applications in optical memory, waveguiding, and formation of micro-optical elements.