Chemistry Reference
In-Depth Information
Figure 3.5 Characteristics of FIONA and
SHRImP. A, theoretical positional uncertainty
(standard deviation) of a single fluorophore vs.
number of photons counted per imaging period
(text equation 3.4). a = pixel size, b = standard
deviation of the background intensity. B, peak
fluorescence intensity from two nearby GFPs
labeling the two heads of a myosin VI molecule,
and displaying two-step photobleaching. C point
spread function (PSF) of the fluorophore pair. D,
PSF of the GFP remaining after the first
photobleaching event. E, the difference between
C and D gives the PSF of the GFP that bleached
first. Positions of the peaks in D and E give the
distance between the fluorophores. Panels B-E
are from Ref. [111].
tainty is due to the
first term under the radical in Equation 3.1, 2% is due to the
second term, and the remainder is due to the third term, the background noise.
Several laboratories have achieved this value of stability in localization at nearly
the theoretical numbers of collected photons [27, 34]. If b increases three-fold,
then the effect of the background becomes more signi
cant (blue curve in
Figure 3.5A). Higher magni
cation and reducing the pixel size further, also
increases
s
m
, because, as the last term in Equation 3.3 shows, the broader spread
of the image over more pixels causes background from those extra pixels to
contribute uncertainty.
The FIONA method has contributed toward understanding the stepping
mechanism of the molecular motors as described later in this chapter (Figures 3.10
and 3.12). Several new methods with entertaining acronyms which have been
derived from FIONA, and have been useful for studying molecular motors
are explained here. Those for general cellular imaging are mentioned at the end
of the chapter.
