Quantitative light imaging of intracelluar transport

Wang, Ru

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https://hdl.handle.net/2142/46934 Copy

Description

Title

Quantitative light imaging of intracelluar transport

Author(s)

Wang, Ru

Issue Date

2014-01-16T18:26:51Z

Director of Research (if dissertation) or Advisor (if thesis)

Popescu, Gabriel

Doctoral Committee Chair(s)

Hsia, K. Jimmy

Committee Member(s)

• Popescu, Gabriel
• Carney, Paul S.
• Toussaint, Kimani C.

Department of Study

Mechanical Sci & Engineering

Discipline

Mechanical Engineering

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Microscopy
• Imaging
• Intracelluar Transport

Abstract

The interior of a living cell is a busy place. Just as understanding the flow of traffic is essential for probing the economy of a major city, exploring the intracellular traffic patterns of cells is fundamental to elucidating their activity. We examine intracellular traffic patterns using a new application of spatial light interference microscopy (SLIM) (Chapter 1). We used this quantitative phase imaging method to measure the dispersion relation, i.e. decay rate vs. spatial mode, associated with mass transport in live cells. This approach, Dispersion-relation Phase Spectroscopy (DPS), (Chapter 3), applies equally well to both discrete and continuous mass distributions without the need for particle tracking. From the quadratic experimental curve specific to diffusion, we extracted the diffusion coefficient as the only fitting parameter. The linear portion of the dispersion relation reveals the deterministic component of the intracellular transport. Our data show a universal behavior where the intracellular transport is diffusive at small scales and deterministic at large scales. We further applied this method to studying transport in neurons (Chapter 4). By modifying a traditional phase contrast microscope, we are able to use SLIM to map the changes in index of refraction across the neuron and its extended processes. What we found was that in dendrites and axons, the transport is mostly active, i.e., diffusion is subdominant. Due to its ability to study specifically labeled structures, fluorescence microscopy is the most widely used technique to study live cell dynamics and function. Fluorescence correlation spectroscopy is an established method for studying molecular transport and diffusion coefficients at a fixed spatial scale. We propose a new approach, dispersion-relation fluorescence spectroscopy (DFS) (Chapter 5), to study the transport dynamics over a broad range of spatial and temporal scales. The molecules of interest are labeled with a fluorophore whose motion gives rise to spontaneous fluorescence intensity fluctuations that are analyzed to quantify the governing mass transport dynamics. These data are characterized by the effective dispersion relation. We report on experiments demonstrating that DFS can distinguish diffusive from advection motion in a model system, where we obtain quantitatively accurate values of both diffusivities and advection velocities. Due to its spatially-resolved information, DFS can distinguish between directed and diffusive transport in living cells. Our data indicate that the fluorescently labeled actin cytoskeleton exhibits active transport motion along a direction parallel to the fibers and diffusive on the perpendicular direction. And we further, for the first time, employed DFS in studying biological structures, for example actin filaments and microtubules (Chapter 6). Our study suggested that dispersion-relation enables to quantify both the spatial and temporal behavior of the transport phenomenon of cytoskeleton with the aid of fluorescence tag. More importantly, in addition to single cellular component specificity, multiple components can be studied simultaneously as long as they are properly labeled. This ability makes the investigation of their interaction possible and our results did show their strong interplay. Apart from the major focus of this thesis, i.e. in-plane mass transport motion (Chapter 3-6), we also took some efforts to study the other different motion, out-of-plane membrane fluctuations of red blood cells (Chapter 2). We present optical measurements of nanoscale red blood cell fluctuations obtained by highly sensitive quantitative phase imaging. These spatio-temporal fluctuations are modeled in terms of the bulk viscoelastic response of the cell. Relating the displacement distribution to the storage and loss moduli of the bulk has the advantage of incorporating all geometric and cortical effects into a single effective medium behavior. The results on normal cells indicate that the viscous modulus is much larger than the elastic one throughout the entire frequency range covered by the measurement, indicating fluid behavior.

Graduation Semester

2013-12

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http://hdl.handle.net/2142/46934

Copyright and License Information

Copyright 2013 Ru Wang

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