I feel "Delta", as the authors called, is much easier to understand than those super-resolution microscopy created by physicists, and definitely much more feasible.
If I understand correctly, it use
* 100x 1.4 NA objective (not the 1.5 one)
* Z section (not the piezoelectric Z-stage)
* ORCA CCD (not EM-CCD)
* not simultaneous capture of the two channel (not the Dual-View attachment from MAG Bio-systems)
* almost any spinning disk microscope can do "Delta"
* use averaging to automatic correct the chromatic aberration
* lsqcurvefit in matlab for data fitting
* ttest2 in matlab for statistic analysis
* I might miss some innovations ...
This method was described in the following paper.
Cell. 2009 May 15;137(4):672-84.
Protein architecture of the human kinetochore microtubule attachment site.
Wan X, O'Quinn RP, Pierce HL, Joglekar AP, Gall WE, DeLuca JG, Carroll CW, Liu ST, Yen TJ, McEwen BF, Stukenberg PT, Desai A, Salmon ED.
Department of Biology, University of North Carolina, Chapel Hill, Chapel Hill, NC 27599, USA.
Chromosome segregation requires assembly of kinetochores on centromeric chromatin to mediate interactions with spindle microtubules and control cell-cycle progression. To elucidate the protein architecture of human kinetochores, we developed a two-color fluorescence light microscopy method that measures average label separation, Delta, at <5 nm accuracy. Delta analysis of 16 proteins representing core structural complexes spanning the centromeric chromatin-microtubule interface, when correlated with mechanical states of spindle-attached kinetochores, provided a nanometer-scale map of protein position and mechanical properties of protein linkages. Treatment with taxol, which suppresses microtubule dynamics and activates the spindle checkpoint, revealed a specific switch in kinetochore architecture. Cumulatively, Delta analysis revealed that compliant linkages are restricted to the proximity of chromatin, suggested a model for how the KMN (KNL1/Mis12 complex/Ndc80 complex) network provides microtubule attachment and generates pulling forces from depolymerization, and identified an intrakinetochore molecular switch that may function in controlling checkpoint activity.
PMID: 19450515
What I learn from the "Delta" method is that super-high resolution microscopy is great, but with appropriate statistics and enough data points, fluorescent protein localization with sub-diffraction accuracy can be obtained using common spinning disk microscopy.
This works because the localization of the fluorescent labels on each protein component is consistent relative to the other protein components. If the underlying labeling distribution and protein orientation was not so constrained, you would need to use "true" super-resolution techniques.
Thanks Andrew. You are correct. Delta approach might not be able to apply to other complexes, i.e. focal adhesion complex