Subcellular Nanoparticle Size Distributions from Light Transmission Spectroscopy

The cell cytoplasm consists of a diverse, complex, self-organized system of objects spanning a broad range of both size and number density. Cells must arrange these active components into a spatially and functionally efficient system, making the comprehensive study of intracellular organization an intriguing and challenging topic. A description of the physical and organizational properties of the cytoplasm can be realized from measurements of the particle size distribution (PSD), or the number density (N) of objects as a function of particle size (D). However, extracted cell contents (lysates) prove to be a distinct measurement challenge owing to the breadth in both size and number density of the subcellular structures and the presence of very small particles. This dissertation presents the development and use of a unique methodology for this task: Light Transmission Spectroscopy (LTS). LTS measures wavelength-dependent light transmittance through an aqueous sample, providing the size and absolute number density of particles in suspension. Significant development of this technique is presented, including the expansion to quantitative LTS (qLTS) where protein and nucleic acid concentrations can be quantified directly from light extinction spectra. With these developments and the use of sample fractionation, LTS was able to provide a PSD for both human (oral, both normal and cancer) and plant (spinach) cells across a broad size range: ~5 nm to ~5 μm. This constitutes the first comprehensive PSD survey of cellular interiors spanning nearly three orders of magnitude in particle size. Power law behavior, where , was observed in the PSD of both cell types, with average power law exponents of 3.04 ± 0.06 for normal human oral cells, 2.84 ± 0.06 for cancer human oral cells, and 2.96 ± 0.08 for spinach cells. This implies that cells exhibit fractal self-similarity. Comparison to common sphere packing models also suggests that cells approach constant-volume packing behavior in their organization. Distinct differences were found between normal and cancer cells including smaller values for the power law exponent, the total volume fraction of particles, and the RNA content in the cancer cells.