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- Results
- Microtubule organization
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insight on the function of MAP65 proteins (Tulin et al., 2012; Portran et al., 2013; Stoppin- 83 Mellet et al., 2013), kinesin motors (Song et al., 1997), katanin-mediated microtubule severing 84 (Stoppin-Mellet et al., 1997) and microtubule dynamics (Moore et al., 1997). However, it is 85 explicitly acknowledged that such in vitro assays should be addressed in biologically coherent 86 systems with physiological relevance of microtubule dynamics (e.g., see Zanic et al., 2013 and 87 discussion in Gardner et al., 2013). Thus, an ideal approach would be to address microtubule 88 dynamics in the complex cellular environment at spatiotemporal resolutions achieved by in vitro 89 assays.
90 Subdiffraction optical microscopy techniques allow subcellular observations below Abbe’s 91 resolution threshold (Verdaasdonk et al., 2014), circumventing the need for TEM. Such 92 approaches permit dynamic subcellular tracking of appropriately tagged structures within living 93 cells (Tiwari & Nagai, 2013). Practically two superresolution strategies exist. The first involves 94 patterned light illumination, allowing superresolution acquisitions by two fundamentally 95 different methods, i.e., stimulated emission-depletion (STED; Hell, 2007) and structured 96 illumination microscopy (SIM; Gustaffson, 2000). The second interrogates the precision of 97 fluorophore localization and includes stochastic optical reconstruction microscopy (STORM; 98 Kamiyama & Huang, 2012) and photoactivation localization microscopy (PALM; Sengupta et 99 al., 2012). The above regimes differ in translational and axial resolution and their temporal 100 efficiency depends on the size of the imaged area. SIM is probably the best compromise for 101 superresolution live imaging as it offers reasonable lateral resolution (ca. 100 nm; Gustaffson, 102 2000) which may be reduced to 50 nm (Rego et al., 2012) and sufficient depth of imaging 103 combined with a reasonable axial resolution (ca. 200 nm). SIM allows dynamic imaging in a 104 broader field of view than STED, at biologically meaningful rates compared to PALM and 105 STORM (Kner et al., 2009) and with deeper imaging capacity compared to other superresolution 106 regimes and to TIRFM/ VAEM (Leung & Chou, 2011). Superresolution approaches have 107 received limited attention in plant cell biology field (Fitzgibbon et al., 2010, Kleine-Vehn et al., 108 2011) and their resolution potential during live imaging was not quantified so far. 109 Hereby, high numerical aperture (NA) objectives are combined with SIM for acquisition and 110 systematic quantification of subdiffraction details of GFP-MBD (Marc et al., 1998; GFP fused to 111
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the microtubule binding domain of mammalian MAP4) and GFP-TUA6-labeled (Shaw et al., 112 2003) cortical microtubules. For such studies, wild type plants and a mitogen activated protein 113
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and underphosphorylation of MAP65-1 (Beck et al., 2010) were used. 115
Results 116
General remarks on sample preparation 117
To optimize SIM imaging of Arabidopsis thaliana hypocotyl epidermal cells, seedlings were 118
grown in darkness since etiolation promotes the thinning of the outer epidermal wall and reduces 119
the thickness of the cuticular surface (Kutschera, 2008). Thus hypocotyl surfaces can be wetted 120
well with acqueous mounting media used for in vivo imaging (1/2 MS medium was used here), 121
excluding air pockets that may introduce additional refractive index mismatches upon imaging. 122
Meanwhile the contour of the etiolated hypocotyl surface is smoother allowing the wider 123
exposure of hypocotyl epidermal cell surfaces underneath the coverslip, as required for high 124
numerical aperture objective with small working distance used hereby. Refractive index 125
mismatches pertaining to fluctuations in coverslip thickness were alleviated by using high 126
performance and low thickness tolerance coverslips. In addition, objective with high numerical 127
aperture (NA 1.57) was used in combination with immersion oil of high refractive index (1.66) 128
in order to obtain maximal possible resolution during SIM, WF, CLSM, TIRF and SD imaging. 129
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Microtubule organization 131
Since details of microtubule organization have been reported using other microscopies, the first 132
task was to characterize qualitatively and quantitatively the resolution potential of SIM and 133
provide relevant comparisons with WF and CLSM with respect to structural features of 134
microtubular arrays. This was particularly important as relevant literature is largely devoid of 135
quantitative resolution data using the above microscopies. 136
In this respect SIM revealed loop-like defects in GFP-MBD cortical microtubule bundles (Fig. 137
1A; top inset and Fig. S1A) which were indiscernible by WF (Fig 1A; middle inset and Fig. 138
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