Holographic Phase Contrast Micro Cytometry

We propose to build a prototype setup for Holographic Phase Contrast Micro Cytometry (HPCMC), which is a unique method developed by us that enables high-throughput measurements of size, shape, optical properties and motion of micron-sized objects, e.g. colloids, bacteria and cells investigated in food and life sciences and related industries. Holographic Phase Contrast Micro Cytometry is a ground-breaking label-free microscopy/cytometry technique that provides statistics of physical properties of individual microparticles and microorganisms in dispersion. Our technique is based on transmission light microscopy. Instead of using light focused in the sample, we shine a collimated laser beam, similar to a laser pointer, through the sample. Then we record so-called in-line holograms that are just the interference between scattered light and the rest of the illuminating beam. To analyse the data, we ‘back propagate’ the light to obtain the full 3D light-field which provides both information on the intensity and the relative phase at every depth. By combining this phase and intensity information we can generate phase contrast images of all the objects throughout the sample. Using our unique and proprietary algorithms, we can characterize all the objects in the sample in terms of the amount of light they scattered, their shape, orientation and so forth. In addition, we can determine dynamic properties from 3D trajectories. HPCMC moreover combines this unique characterization power with a throughput that is similar to that of flow cytometry (FC). Flow cytometry also measures signals from individual microparticles and microorganisms, but these have to be previously separated, typically by individual encapsulation in drops. Crucially, flow cytometry relies heavily on labeling the samples with fluorescence markers and combining this with analysis of the light scattered from the object. Thus, samples are subjected to invasive pre-labeling and no image is acquired. The latter means that decisive properties such as shape and size cannot be determined. There is also no information acquired regarding dynamic information of the object, such as its mobility (diffusion, rotation, propulsion, etc.), which are critical parameters in life science but unavailable using high-throughput methods today. HPCMC provides quantitative measurements of physical properties including mobility of individual objects at high acquisition rates and thus accurate statistics of entire sample populations with sub-population variations. In contrast to whole population-based methods such as standard scattering techniques, HPCMC can therefore refine datasets to identify sub-populations with distinguishing physical or motile properties within a population and thereby open new doors to investigators and in quality control.