Digital holographic microscopy allows determination of dynamic changes in the optical thickness profile of a transparent object with subwavelength accuracy. Here, we report a quantitative phase laser microsurgery system for evaluation of cellular/ sub-cellular dynamic changes during laser micro-dissection. The proposed method takes advantage of the precise optical manipulation by the laser microbeam and quantitative phase imaging by digital holographic microscopy with high spatial and temporal resolution. This system will permit quantitative evaluation of the damage and/or the repair of the cell or cell organelles in real time.

The ability to inject exogenous material as well as to alter subcellular structures in a minimally invasive manner using a laser microbeam has been useful for cell biologists to study the structure-function relationship in complex biological systems. We describe a quantitative phase laser microsurgery system, which takes advantage of the combination of laser microirradiation and short-coherence interference microscopy. Using this method, quantitative phase images and the dynamic changes of phase during the process of laser microsurgery of red blood cells (RBCs) can be evaluated in real time. This system would enable absolute quantitation of localized alteration/damage to transparent phase objects, such as the cell membrane or intracellular structures, being exposed to the laser microbeam. Such quantitation was not possible using conventional phase-contrast microscopy.

We demonstrate the formation of low-and high-density plasmas in aqueous media using sub-nanosecond laser pulses delivered at low numerical aperture (NA ¼ 0.25). We observe two distinct regimes of plasma formation in deionized water, phosphate buffered saline, Minimum Essential Medium (MEM), and MEM supplemented with phenol red. Optical breakdown is first initiated in a low-energy regime and characterized by bubble formation without plasma luminescence with threshold pulse energies in the range of E p % 4-5 lJ, depending on media formulation. The onset of this regime occurs over a very narrow interval of pulse energies and produces small bubbles (R max ¼ 2-20 lm) due to a tiny conversion (g < 0.01%) of laser energy to bubble energy E B . The lack of visible plasma luminescence, sharp energy onset, and low bubble energy conversion are all hallmarks of low-density plasma (LDP) formation. At higher pulse energies (E p ¼ 11-20 lJ), the process transitions to a second regime characterized by plasma luminescence and large bubble formation. Bubbles formed in this regime are 1-2 orders of magnitude larger in size ðR max տ 100 lmÞ due to a roughly two-order-of-magnitude increase in bubble energy conversion (g տ 3%). These characteristics are consistent with high-density plasma formation produced by avalanche ionization and thermal runaway. Additionally, we show that supplementation of MEM with fetal bovine serum (FBS) limits optical breakdown to this high-energy regime. The ability to produce LDPs using sub-nanosecond pulses focused at low NA in a variety of cell culture media formulations without FBS can provide for cellular manipulation at high throughput with precision approaching that of femtosecond pulses delivered at high NA. V C 2014 AIP Publishing LLC.