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Applications of spatial frequency modulation for imaging in cell deformation cytometry
Neumann, Jacob A.
Neumann, Jacob A.
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2018
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2019-02-28
Abstract
In this thesis, we develop two novel system architectures for the measurement of the flow position, size, and shape of red blood cells flowing in a microfluidic channel for the primary purpose of cell elasticity cytometry. The current state of the art relies upon the use of expensive high speed (of order 100 fps) CCD cameras to observe optically stretched red blood cell relaxing from a stretched state. This method also requires the use of computationally expensive edge finding techniques in order to convert the images into useful size information, which is then used to compute the cell elasticity. Our designs are fundamentally derived from the technique SPaItial Frequency modulation for Imaging (SPIFI). SPIFI is a microscopy technique prized for its ability to recover one and two dimensional information using a single element detector, such as a photodiode, instead of a camera. By applying a spatially varying frequency modulation to the excitation source, spatial information is encoded in the frequency spectrum of the beam. The light emitted by the microscope objective can subsequently be collected and analyzed through examination of its periodogram. Because each frequency component is mapped to a spatial location, the amplitude of the periodogram can be used to create an image of our specimen. We propose two systems that take advantage of the underlying principle of SPIFI (that higher dimensional information can be collected using a single element detector by using spatial modulation of light). The first uses a static Cartesian coordinate SPIFI mask placed directly above a microfluidic channel. We showed qualitatively that such a system is capable of determining the flow position of a target in a microfluidic flow and its size using computational and experimental methods. Our experiment used a laser beam scanning across a mask as a macroscopic correlate of a fluorescent target flowing beneath a mask. Under a set of restrictions generally met by red blood cells, it is even capable of recovering limited information about the shape of the cell. However, the static mask system is unable to provide reliable shape and size information about the target if its size is changing due to the time-frequency uncertainty principle and the coupling of the target flow speed with frequency and temporal window parameters. High flow speed can cause cell deformation, complicating elasticity measurements. We also demonstrate for the first time that femtosecond laser micromachined masks are capable of modulating light of wavelength 632 nm and 800 nm sufficiently for conventional SPIFI applications, allowing masks to be produced more cheaply and with greater flexibility of configuration. The second system relies on a spinning SPIFI mask, best described in terms of radial coordinates. The frequency and temporal window are entirely controlled by the mask and spin motor properties, allowing it measure the size of a red blood cell relaxing from a stretched state. We show mathematically that the system collapses the two dimensional information of the target into a one dimensional function which is directly recovered by the examining the periodogram of the signal produced by our system. We then show that we can recover shape information from this function. We also show that our model qualitatively matches experimental results using macroscopic opaque targets. Both techniques that we demonstrate require further development which can be accomplished rapidly. However, the spinning mask architecture has the most potential due to its ability to measure cell size as it relaxes from a stretched state. As such, further research on the spinning mask system ought to be prioritized.
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