GPC light shaper for speckle-free one- and two-photon contiguous pattern excitation.

Generalized Phase Contrast (GPC) is an efficient method for generating speckle-free contiguous optical distributions useful in diverse applications such as static beam shaping, optical manipulation and recently, for excitation in two-photon optogenetics. To fully utilize typical Gaussian lasers in such applications, we analytically derive conditions for photon efficient light shaping with GPC. When combined with the conditions for optimal contrast developed in previous works, our analysis further simplifies GPC's implementation. The results of our analysis are applied to practical illumination shapes, such as a circle and different rectangles commonly used in industrial or commercial applications. We also show simple and efficient beam shaping of arbitrary shapes geared towards biophotonics research and other contemporary applications. Optimized GPC configurations consistently give ~84% efficiency and ~3x intensity gain. Assessment of the energy savings when comparing to conventional amplitude masking show that ~93% of typical energy losses are saved with optimized GPC configurations.

Matched filtering Generalized Phase Contrast using binary phase for dynamic spot- and line patterns in biophotonics and structured lighting.

This work discusses the use of matched filtering Generalized Phase Contrast (mGPC) as an efficient and cost-effective beam shaper for applications such as in biophotonics, optical micromanipulation, microscopy and two-photon polymerization. The theoretical foundation of mGPC is described as a combination of Generalized Phase Contrast and phase-only correlation. Such an analysis makes it convenient to optimize an mGPC system for different setup conditions. Results showing binary-only phase generation of dynamic spot arrays and line patterns are presented.

Optical forces through guided light deflections.

Optical trapping and manipulation typically relies on shaping focused light to control the optical force, usually on spherical objects. However, one can also shape the object to control the light deflection arising from the light-matter interaction and, hence, achieve desired optomechanical effects. In this work we look into the object shaping aspect and its potential for controlled optical manipulation. Using a simple bent waveguide as example, our numerical simulations show that the guided deflection of light efficiently converts incident light momentum into optical force with one order-of-magnitude improvement in the efficiency factor relative to a microbead, which is comparable to the improvement expected from orthogonal deflection with a perfect mirror. This improvement is illustrated in proof-of-principle experiments demonstrating the optical manipulation of two-photon polymerized waveguides. Results show that the force on the waveguide exceeds the combined forces on spherical trapping handles. Furthermore, it shows that static illumination can exert a constant force on a moving structure, unlike the position-dependent forces from harmonic potentials in conventional trapping.

BioPhotonics workstation: a versatile setup for simultaneous optical manipulation, heat stress, and intracellular pH measurements of a live yeast cell.

In this study we have modified the BioPhotonics workstation (BWS), which allows for using long working distance objective for optical trapping, to include traditional epi-fluorescence microscopy, using the trapping objectives. We have also added temperature regulation of sample stage, allowing for fast temperature variations while trapping. Using this modified BWS setup, we investigated the internal pH (pH(i)) response and membrane integrity of an optically trapped Saccharomyces cerevisiae cell at 5 mW subject to increasing temperatures. The pH(i) of the cell is obtained from the emission of 5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester, at 435 and 485 nm wavelengths, while the permeability is indicated by the fluorescence of propidium iodide. We present images mapping the pH(i) and permeability of the cell at different temperatures and with enough spatial resolution to localize these attributes within the cell. The combined capability of optical trapping, fluorescence microscopy and temperature regulation offers a versatile tool for biological research.

Intracellular pH distribution as a cell health indicator in Saccharomyces cerevisiae.

Internal pH regulation is vital for many cell functions, including transport mechanisms and metabolic enzyme activity. More specifically, transport mechanisms are to a wide degree governed by internal pH distributions. We introduce the term standard deviation of the intracellular pH (s.d.(pH(int))) to describe the internal pH distributions. The cellular pH distributional response to external stress such as heat has not previously been determined. In this study, the intracellular pH (pH(i)) and the s.d.(pH(int)) of Saccharomyces cerevisiae cells exposed to supralethal temperatures were measured using fluorescence ratio imaging microscopy (FRIM). An exponential decline in pH(i) was observed after an initial small decline. For the first time, we report the use of FRIM for determining in vivo plasma membrane proton permeability coefficients in yeast. Furthermore, the exponential decay of pH(i) and the rupture of the cell plasma membrane, as measured by propidium iodide staining, at 70°C were not simultaneous but were separated by a significant temporal difference. Finally, a nonlinear relationship between the pH(i) and s.d.(pH(int)) was found; i.e. the s.d.(pH(int)) was significantly more sensitive to supralethal temperatures than pH(i). s.d.(pH(int)) is therefore proposed as an early health/vitality indicator in S. cerevisiae cells exposed to heat stress.

Effect of long- and short-term exposure to laser light at 1070 nm on growth of Saccharomyces cerevisiae.

The effect of a 1070-nm continuous and pulsed wave ytterbium fiber laser on the growth of Saccharomyces cerevisiae single cells is investigated over a time span of 4 to 5 h. The cells are subjected to optical traps consisting of two counterpropagating plane wave beams with a uniform flux along the x, y axis. Even at the lowest continuous power investigated-i.e., 0.7 mW-the growth of S. cerevisiae cell clusters is markedly inhibited. The minimum power required to successfully trap single S. cerevisiae cells in three dimensions is estimated to be 3.5 mW. No threshold power for the photodamage, but instead a continuous response to the increased accumulated dose is found in the regime investigated from 0.7 to 2.6 mW. Furthermore, by keeping the delivered dose constant and varying the exposure time and power-i.e. pulsing-we find that the growth of S. cerevisiae cells is increasingly inhibited with increasing power. These results indicate that growth of S. cerevisiae is dependent on both the power as well as the accumulated dose at 1070 nm.

Efficient optical trapping and visualization of silver nanoparticles.

We performed efficient optical trapping combined with sensitive optical detection of individual silver nanoparticles. The particles ranging in size from 20 to 275 nm in diameter were trapped in three dimensions using low laser power by minimizing spherical aberrations at the focus. The optical forces were quantified, and we found that the larger the particle, the stronger the optical force. The particles were imaged by an additional strongly scattered laser.