The positioning accuracy when a phase-only one dimensional spatial light modulator (SLM) is used for beam
steering is limited by the number of pixels and their quantized phase modulation. Optimizing the setting of the
SLM pixels individually can lead to the inaccuracy being a significant fraction of the diffraction limited spot size.
This anomalous behaviour was simulated numerically, and experiments showed the same phenomena with very
good agreement. However, by including an extra degree of freedom in the optimization of the SLM setting, we
show that the accuracy can be improved by a factor proportional to the number of pixels in the SLM.
Optical manipulation techniques have become an important research tool for single cell experiments in microbiology.
Using optical tweezers, single cells can be trapped and held during long experiments without risk of cross contamination
or compromising viability. However, it is often desirable to not only control the position of a cell, but also to control its
environment. We have developed a method that combines optical tweezers with a microfluidic device. The microfluidic
system is fabricated by soft lithography in which a constant flow is established by a syringe pump. In the microfluidic
system multiple laminar flows of different media are combined into a single channel, where the fluid streams couple
viscously. Adjacent media will mix only by diffusion, and consequently two different environments will be separated by
a mixing region a few tens of micrometers wide. Thus, by moving optically trapped cells from one medium to another
we are able to change the local environment of the cells in a fraction of a second. The time needed to establish a change
in environment depends on several factors such as the strength of the optical traps and the steepness of the concentration
gradient in the mixing region. By introducing dynamic holographic optical tweezers several cells can be trapped and
analyzed simultaneously, thus shortening data acquisition time. The power of this system is demonstrated on yeast
(<i>Saccharomyces cerevisiae</i>) subjected to osmotic stress, where the volume of the yeast cell and the spatial localization of
green fluorescent proteins (GFP) are monitored using fluorescence microscopy.
In recent years there has been a growing interest in the use of optical manipulation techniques, such as optical
tweezers, in biological research as the full potential of such applications are being realized. Biological research is
developing towards the study of single entities to reveal new behaviors that cannot be discovered with more
traditional ensemble techniques. To be able to study single cells we have developed a new method where a
combination of micro-fluidics and optical tweezers was used. Micro-fluidic channels were fabricated using soft
lithography. The channels consisted of a Y-shaped junction were two channels merged into one. By flowing
different media in the two channels in laminar flow we were able to create a sharp concentration gradient at the
junction. Single cells were trapped by the tweezers and the micro-fluidic system allowed fast environmental
changes to be made for the cell in a reversible manner. The time required to change the surroundings of the cell
was limited to how sharp mixing region the system could create, thus how far the cells had to be moved using
the optical tweezers. With this new technique cellular response in single cells upon fast environmental changes
could be investigated in real time. The cellular response was detected by monitoring variations in the cell by
following the localization of fluorescently tagged proteins within the cell.
We explore the Generalized Phase Contrast (GPC) approach for optical sorting in microfluidic systems. A microsystem is used in which two streams meet, interact and separate in an X-shaped channel. When the flow in the two arms of the X is balanced, the laminar flow that exists at very low Reynolds numbers ensures minimal stream blending and the fluid separates without mixing (i.e. diffusion is negligible). Optical forces due to an intensity pattern can be fashioned to induce a selective deflection of particles between the two streams. This method is known as optical fractionation (OF). In brief, OF uses the same mechanisms as optical tweezers to exert forces upon microscopic particles. OF has been shown to have an exponential size selectivity. This means that the interaction between the streams can be made to discriminate by particle size at a critical flow velocity. With correctly adjusted flow velocity, particles with a certain size will more often shift to the other stream than another particle size. One method for creating the light pattern is by interference of several beams that are variably attenuated using mechanical means. However, this approach offers low
optical efficiency and is not easily reconfigured. The GPC method offers a solution that gives the possibility to instantaneously reconfigure the intensity pattern by a method that is inherently computer-controllable. This enables one to rapidly test various intensity patterns to optimize sorting of particles.