Angular scattering can be used to obtain morphological information from biological specimen, such as the mean size of organelles. We will investigate the limitations that arise when fitting cell scattering to a Mie theory model and extracting organelle size estimates. Using simulation studies of the scattering of two organelle populations in a cell (mitochondria and lysosomes), we will determine under what conditions multiple size distribution parameters can be estimated. Additionally, the analysis method that will allow for the most accurate and smallest organelle size changes to be measured will be investigated computationally.
Angularly resolved elastic light scattering is an established technique for probing the average size of organelles in biological tissue and cellular ensembles. Focusing of the incident light to illuminate no more than one cell at a time restricts the minimum forward-scattering angle θmin that can be detected. Series of simulated single-cell angular-scattering patterns have been generated to explore how size estimates vary as a function of θmin. At a setting of θmin = 20 deg, the size estimates hop unstably between multiple minima in the solution space as simulated noise (mimicking experimentally observed levels) is varied. As θmin is reduced from 20 deg to 10 deg, the instability vanishes, and the variance of estimates near the correct answer also decreases. The simulations thus suggest that robust Mie theory fits to single-cell scattering at 785 nm excitation require measurements down to at least 15 deg. Notably, no such instability was observed at θmin = 20 deg for narrow bead distributions. Accurate sizing of traditional calibration beads is, therefore, insufficient proof that an angular-scattering system is capable of robust analysis of single cells. Experimental support for the simulation results is also presented using measurements on cells fixed with formaldehyde.
Functional near-infrared spectroscopy (fNIRS) research to date has tended to publish group-averaged rather than individual infant data due to normative basic research goals. Acquisition of individual infant time courses holds interest, however, both for cognitive science and particularly for clinical applications. Infants are more difficult to study than adults as they cannot be instructed to remain still. In addressing this, upright infants pose several associated complications for the researcher. We identified and optimized the factors that affect the quality of fNIRS data from individual 6- to 9-month-old infants exposed to a visual stimulation paradigm. The fNIRS headpiece was reconfigured to reduce inertia, increase comfort, and improve conformity to the head, while preserving fiber density to avoid missing the visual cortex activation. The visual-stimulation protocol was modified to keep the attention of infants throughout the measurement, thus helping to reduce motion artifacts. Adequate optical contact was verified by checking power levels before each measurement. By revising our experimental process and our data rejection criteria to prioritize good optical contact, we report for the first time usable hemodynamic data from 83% of infants and that two-thirds of infants produced a statistically significant fNIRS response.
The literature contains several reports of Mie-like fits to angular-domain elastic scattering measurements from multiple cells or isolated mitochondria. In these studies, the sampling volume typically contains hundreds or thousands of mitochondria, allowing for the size distribution of mitochondria to be modeled as a smooth function, (e.g. Gaussian or log-normal) with a small number of free parameters. In the case of a single-cell volume containing significantly fewer mitochondria, the true size distribution will no longer be as smooth. Increasing the number of free parameters can lead to unstable fits, however, as the forward-directed angular scattering pattern from such a population illuminated with 785 nm light is a monotonically decaying radial function with few distinct features. Using simulations, we have investigated the limitations of modeling single-cell mitochondrial scattering using smooth population distributions of Mie scatterers. In different instances, the fidelity of the estimated size information can be limited by the number of organelles, the angular detection range, or the non-ideality of the data (both speckle and shot noise). We will describe the conditions under which each of these effects dominates. We will also discuss whether mean and standard deviation are the best sizes to report from such Mie modeling, or if there are other size parameters that have greater fidelity to the true, non-smooth size distributions.
KEYWORDS: Scattering, Mie scattering, Scatter measurement, Calibration, Cancer, Time metrology, Digital imaging, Biological research, Tissues, Current controlled current source
The goal of this project is to estimate non-nuclear organelle size distributions in single cells by measuring angular scattering patterns and fitting them with Mie theory. Simulations have indicated that the large relative size distribution of organelles (mean:width≈2) leads to unstable Mie fits unless scattering is collected at polar angles less than 20 degrees. Our optical system has therefore been modified to collect angles down to 10 degrees. Initial validations will be performed on polystyrene bead populations whose size distributions resemble those of cell organelles. Unlike with the narrow bead distributions that are often used for calibration, we expect to see an order-of-magnitude improvement in the stability of the size estimates as the minimum angle decreases from 20 to 10 degrees. Scattering patterns will then be acquired and analyzed from single cells (EMT6 mouse cancer cells), both fixed and live, at multiple time points. Fixed cells, with no changes in organelle sizes over time, will be measured to determine the fluctuation level in estimated size distribution due to measurement imperfections alone. Subsequent measurements on live cells will determine whether there is a higher level of fluctuation that could be attributed to dynamic changes in organelle size. Studies on unperturbed cells are precursors to ones in which the effects of exogenous agents are monitored over time.
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