Using Spectral Phasor Analysis of UV-Excited Autofluorescence to Reveal Small Differences between Chemically-Induced Mitochondrial Responses

Abstract

Microtubules are hollow protein filaments consisting of the alpha/beta-tubulin subunit, and they play important roles in various biological processes such as cell division, intracellular transport, cell motility and cell morphogenesis. The dynamics of microtubules is critical to the proper function of microtubules in cell division. One of the challenges in improving our understanding of microtubule dynamics is the small size of tubulin subunits. Because each subunit is only few nanometers in size and significantly smaller than the wavelength of light, optical microscopy cannot be used to resolve the interactions of the subunits, which lead to the formation of microtubules, in real time. We developed an in vitro spectroscopy assay for detecting microtubule formation below the diffraction limit of light. The assay is based on Förster resonance energy transfer between fluorescent molecules of a single type (homoFRET). Our results indicate that homoFRET can be used to detect short microtubules even when they are diffraction limited (smaller than few hundred nanometers). We also demonstrate that when fluorophores with appropriate Förster distance are used, this technique can be highly sensitive to the formation of microtubules but less sensitive to the extent of microtubule elongation, making it suitable for detecting microtubule nucleation.