Probing microtubules polarity in mitotic spindles in situ using Interferometric Second Harmonic Generation Microscopy

Abstract

The polarity of microtubules is thought to be involved in spindle assembly, cytokinesis or active molecular transport. However, its exact role remains poorly understood, mainly because of the challenge to measure microtubule polarity in intact cells. We report here the use of fast Interferometric Second Harmonic Generation microscopy to study the polarity of microtubules forming the mitotic spindles in a zebrafish embryo. This technique provides a powerful tool to study mitotic spindle formation and may be directly transferable for investigating the kinetics and function of microtubule polarity in other aspects of subcellular motility or in native tissues.

Molecular understanding of label-free second harmonic imaging of microtubules

“…The current understanding of how neuronal fibers generate detectable SH signals is limited, and boils down to the concept that the SH phenomenon is only present in case of uniform polarity of microtubules in axons, mature dendrites, and mitotic spindles in non-neuronal cells 24–27,38–40 . However, we show that, in case sufficient detection sensitivity is available, SHG imaging of non-neuronal cells such as fibroblasts is not only limited to detecting microtubules in the spindle apparatus during cell division.…”

DANPY (dimethylaminonaphthylpyridinium): an economical and biocompatible fluorophore

“…6 Because SHG can also be generated by chiral well-ordered structures such as collagen and myosin, even cultured cells can occasionally show "auto-SHG" in the absence of a dye. 95,96 The source of the signal in the nucleus of the cell highlighted with the blue arrows in Fig. 9 is inconclusive and could be SHG from DANPY-1 bound to DNA or auto-SHG from microtubules of the mitotic spindle.…”

Harmonic Generation Microscopy 2.0: New Tricks Empowering Intravital Imaging for Neuroscience

“…Collagen fibrils are a well-known example representing an extracellular pseudocrystalline phase studied by SHG (Roth and Freund, 1979, 1982; Freund et al, 1986). By contrast, an intracellular phase is found in the cytoskeletal element of microtubules from which SHG has been detected in the context of mitotic spindles (Campagnola et al, 2002; Bancelin et al, 2017) and axons (Dombeck et al, 2003; Kwan et al, 2008; Lim and Danias, 2012a; Psilodimitrakopoulos et al, 2012; Van Steenbergen et al, 2019). The unique phase of microtubules, i.e., uniform polarity, is a defining characteristic of axons as opposed to dendrites (Baas et al, 1988), rendering the SHG signal specific to the axons (Figures 1A–C).…”