Abstract
.We demonstrate the use of wide-field high-throughput second-harmonic (SH) microscopy for investigating cytoskeletal morphological changes on the single-cell level. The method allows for real-time, in vitro, label-free measurements of cytoskeletal changes that can, under certain conditions, be quantified in terms of orientational distribution or in terms of changes in the number of microtubules. As SH generation is intrinsically sensitive to noncentrosymmetrically structured microtubules, but not to isotropic or centrosymmetric materials, we use it to probe the microtubule structure in the cytoskeleton when it undergoes dynamic changes induced by the application of nocodazole, a well-known microtubule-destabilizing drug that reversibly depolymerizes microtubules. In addition, the orientational directionality of microtubules in neurites and cell bodies is determined label-free using SH polarimetry measurements. Finally, we use spatiotemporal SH imaging to show label-free, real-time nocodazole-induced morphological changes in neurons of different age and in a single axon.
Highlights
Microtubules are highly dynamic polymers[1] that constitute a major component of the cytoskeletal network of neuronal cells
Assuming that the orientational distribution within the axon is unaffected by nocodazole, we convert the SH intensity changes into a change in the number of microtubules present in the structures and show the possibility of our imaging technique to image cytoskeletal structural changes label-free
Wide-field, high-throughput SH microscopy was applied to study living mammalian neurons with the aim of demonstrating the possibility to extract the orientational distribution of microtubules as well as obtaining dynamic label-free spatiotemporal stability information in vitro
Summary
Microtubules are highly dynamic polymers[1] that constitute a major component of the cytoskeletal network of neuronal cells. This quadratic dependence is referred to as spatial coherence This means that axons that have microtubules organized unidirectionally will emit a much stronger SH intensity than structures with a bidirectional orientation of microtubules, allowing for identification of the axon by means of the intensity.[37] in principle, SH imaging allows for identification of different morphological parts of the cytoskeleton, and SH polarimetry can be used to determine orientational distributions of noncentrosymmetric structures. We determine the orientational distribution of microtubules and map the relative intensity of different morphological structures and perform in-vitro imaging of the effects of nocodazole on the axon of a corpus of neurons at different stages of maturity. Assuming that the orientational distribution within the axon is unaffected by nocodazole, we convert the SH intensity changes into a change in the number of microtubules present in the structures and show the possibility of our imaging technique to image cytoskeletal structural changes label-free
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