Holographic optical tweezers make it possible to move multiple particles around at the same time, in three dimensions. Held in the focal point of a laser beam, these particles (usually micron-sized and dielectric) will follow the focal spot as it is moved around. Liquid crystal spatial light modulators are an increasingly popular way to steer and refocus these beams using computer generated holograms, which can be generated and displayed in real time for interactive use.
One issue raised by the ability to move things around in 3D is that of imaging: typically, the bright field microscopy that accompanies optical tweezers works only over an axial range of a few microns. Chen et al. get around this by using a volume hologram in the imaging path, which brings different depths of the sample into focus at different points on the camera. While two-dimensional holograms have been used to image at several depths simultaneously in a microscope before, the use of thick gratings is potentially more efficient and less reliant on having monochromatic light in the sample. Their system currently creates images of two planes 50 microns apart, but the group has previously demonstrated gratings capable of working in five planes at the same time.
Volume holograms designed this way produce images of the sample on the camera, and thus require very little extra processing in order to be useful to the operator. This is a distinct advantage over digital holographic microscopy, for example, where computationally expensive reconstruction algorithms can mean that particles cannot be followed in real time. Computational overheads are particularly important when high-speed cameras are employed to do particle tracking at hundreds or thousands of frames per second. Such high-speed tracking is commonly employed to measure forces and displacements, and tracking on-the-fly removes the need to store huge volumes of data for later off-line analysis. Another potential advantage of volume holograms is that their angle (and focus) sensitivity can be exploited to direct most of the light from a given depth in the sample into the image where it is in focus. In contrast, a 2D hologram splits the light equally between all focal depths, even ones where it would be very out of focus (which could legitimately be considered a waste of light).
Lastly, the team showed that this system can work well with fluorescence imaging, rather than coherent bright-field illumination as used in most digital holographic microscopes. This makes the technique much more widely applicable to microscopy in general, and the volume hologram might just prove to be a convenient extra filter to slide in when working with samples that have more depth than a 2D camera can cope with. Their work also has a pleasing symmetry: one hologram to control the laser that goes in to form the traps, another to shape the light that comes out carrying the image.
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