Nanometer-localized multiple single-molecule fluorescence microscopy

Qu et al. 10.1073/pnas.0402155101.

Supporting Information

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Supporting Figure 8




Supporting Figure 8

Fig. 8. Shown is an example in which five dyes located within a small region can be resolved and localized. The number of counts at the peak pixel within an 11 × 11 pixel area is plotted versus time. Five photobleaching events are observed, and the average count level before each photobleaching event is marked by an arrow and referred as steps 1-5. The dye photobleached right after step 1 will be called dye 1, and dyes 2 to 5 are named similarly. Three data points marked (a), (b), and (c) are examples of data that need to be excluded from the standard NALMS data analysis procedure. Point a is an example in which dye 1 photobleaches during the signal integration of one frame. Such data points are excluded from the analysis because they bias the dye emission intensity estimation. Point b is an example in which dye 2 is observed to be transiently in a totally "off" state for one frame so the count level drops to the level of Step 3. Point c is an example in which a photobleached dye briefly comes back to an "on" state and gives out some photons. Such discrete off and on states are excluded from the data analysis and do not affect the localization result.(Inset) The relative location of the dyes determined by NALMS position analysis and the SD of the measured distance is determined with Eqs. 3 and 4.

This Article

  1. Published online before print July 26, 2004, doi: 10.1073/pnas.0402155101 PNAS August 3, 2004 vol. 101 no. 31 11298-11303
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