On probing conformation of microtubules by second-harmonic generation

Sharoukhov, Denis; Lim, Henry C.

doi:10.1080/09500340.2015.1080866

Cited by 7 publications

(7 citation statements)

References 46 publications

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“…Laser scanning microscopy techniques that exploit nonlinear optical effects such as two‐photon excited fluorescence and second harmonic generation (SHG) have been proven to be useful tools in biomedical tissue imaging . SHG is a label‐free, coherent second order nonlinear investigation method which was successfully used to date for imaging collagen , myosin and tubulin .…”

Section: Introductionmentioning

confidence: 99%

Improved quantification of collagen anisotropy with polarization‐resolved second harmonic generation microscopy

Hristu

Stanciu

Trancă

2016

Journal of Biophotonics

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Imaging tissue samples by polarization-resolved second harmonic generation microscopy provides both qualitative and quantitative insights into collagen organization in a label-free manner. Polarization-resolved second harmonic generation microscopy goes beyond simple intensity-based imaging by adding the laser beam polarization component and applying different quantitative metrics such as the anisotropy factor. It thus provides valuable information on collagen arrangement not available with intensity measurements alone. Current established approaches are limited to calculating the anisotropy factor for only a particular laser beam polarization and no general guidelines on how to select the best laser beam polarization have yet been defined. Here, we introduce a novel methodology for selecting the optimal laser beam polarization for characterizing tissues using the anisotropy in the purpose of identifying cancer signatures. We show that the anisotropy factor exhibits a similar laser beam polarization dependence to the second harmonic intensity and we combine it with the collagen orientation index computed by Fast Fourier Transform analysis of the recorded images to establish a framework for choosing the laser beam polarization that is optimal for an accurate interpretation of polarization-resolved second harmonic generation microscopy images and anisotropy maps, and hence a better differentiation between healthy and dysplastic areas. SHG image of skin tissue (a) and a selected area of interest for which we compute the SHG intensity (b) and anisotropy factor (c) dependence on the laser beam polarization and also the FFT spectrum (d) to evaluate the collagen orientation index.

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Section: Introductionmentioning

confidence: 99%

Improved quantification of collagen anisotropy with polarization‐resolved second harmonic generation microscopy

Hristu

Stanciu

Trancă

2016

Journal of Biophotonics

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“…We tested pSHG for measuring allosteric dynamics upon binding with taxol. The pSHG profile of microtubules was much different from that of collagen (Figures 1D,E) (Odin et al, 2009; Psilodimitrakopoulos et al, 2012, 2013; Sharoukhov and Lim, 2016), but the overall effect of taxol was too small to resolve (Sharoukhov and Lim, 2016). Recently, the conformational change upon binding to kinesin KIF5C has been investigated using pSHG and microtubules ex vivo from Xenopus laevis egg extract, and the data suggests considerable allostery of microtubules (Shima et al, 2018) (Figure 1F).…”

Section: Peptide Bonds As a Source Of Endogenous Shgmentioning

confidence: 99%

“…Dashed lines, characteristic sectorial degeneration of glaucoma (redrawn from Sharoukhov et al, 2018). (D,E) SHG intensity for collagen (D) and microtubules (E) , respectively, and SHG polarization anisotropy (pSHG) corresponding to the dashed lines as a function of the polarization angle (redrawn from Sharoukhov and Lim, 2016). (F) SHG anisotropy of microtubule changes upon KIF5C binding.…”

Section: Peptide Bonds As a Source Of Endogenous Shgmentioning

confidence: 99%

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

Lim

2019

Front. Mol. Biosci.

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Optical harmonic generation, e.g., second- (SHG) and third-harmonic generation (THG), provides intrinsic contrasts for three-dimensional intravital microscopy. Contrary to two-photon excited fluorescence (TPEF), however, they have found relatively specialized applications, such as imaging collagenous and non-specific tissues, respectively. Here we review recent advances that broaden the capacity of SHG and THG for imaging the central nervous system in particular. The fundamental contrast mechanisms are reviewed as they encode novel information including molecular origin, spectroscopy, functional probes, and image analysis, which lay foundations for promising future applications in neuroscience.

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“…Based on the cylindrical symmetry of the system, one assumes spatial isotropy around two angles ðψ; ϕÞ and directionally around only one orientational angle (θ). For a nonresonant optical process (involving only virtual energy states), there are only two nonzero [31][32][33][34] χ ð2Þ ijk ð2ωÞ tensor elements, namely χ ð2Þ yyy ð2ωÞ and χ ð2Þ yxx ð2ωÞ. Considering this analysis, one can measure the orientational angle of microtubules in a structure by measuring different polarization combinations.…”

Section: Introductionmentioning

confidence: 99%

“…Considering this analysis, one can measure the orientational angle of microtubules in a structure by measuring different polarization combinations. One way to obtain the tilt angle of a microtubule segment at a single pixel is to compute the ratio: [31][32][33][34] E Q -T A R G E T ; t e m p : i n t r a l i n k -; e 0 0 3 ; 6 3 ; 2 7 8 Ið2ωÞ YYY Ið2ωÞ XXX ¼ tan 2 ðθÞ:…”

Section: Introductionmentioning

confidence: 99%

Mapping of real-time morphological changes in the neuronal cytoskeleton with label-free wide-field second-harmonic imaging: a case study of nocodazole

et al. 2019

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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. © The Authors. Published by SPIE under a Creative Commons Attribution 4.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI.

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On probing conformation of microtubules by second-harmonic generation

Cited by 7 publications

References 46 publications

Improved quantification of collagen anisotropy with polarization‐resolved second harmonic generation microscopy

Improved quantification of collagen anisotropy with polarization‐resolved second harmonic generation microscopy

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

Mapping of real-time morphological changes in the neuronal cytoskeleton with label-free wide-field second-harmonic imaging: a case study of nocodazole

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