Biology Reference
In-Depth Information
1.
Each recovery series is checked to ensure that no convective motion of the solu-
tion has occurred. The authors use a subroutine in MPL that determines any lat-
eral displacement of the center of the bleach using a center of mass calculation.
2.
The effects of nonuniformity in illumination and detector response are corrected
by dividing each recovery image by the prebleach image to produce a floating-
point ratio image.
3.
Pixels contained within three bleach widths of the center are quantified and a
normalized radial intensity distribution is determined for each image, with the
bleach center set as the origin (
Fig. 4
). The average intensity at the image periph-
ery (between three and four bleach widths from the center) is set as the mean
background intensity.
4.
Intensity profile data are passed from MPL to Excel. The radial distribution of
bleached fluorophores is determined by subtracting radial intensity values from
the image background intensity.
5.
The variance of the radial distribution of bleached fluorophores is calculated and
plotted as a function of time (
see
ref.
[13]
for background details of how to calcu-
late the variance values). For well-defined populations of monomeric diffusants,
a linear relationship exists between variance and time. The lateral translational
diffusion coefficient is calculated from the gradient determined from a linear
least-squares fit to the data.
6.
A nonlinear dependence of variance upon time can result from either an underes-
timate of the apparent background intensity as photobleached components reach
the edge of the field of view, or, the presence of multiple independently diffusing
components. The first problem is rectified by excluding data from longer time-
points. Multiple components having greatly differing diffusion coefficients show
a characteristic multicomponent recovery of total intensity in the bleached area.
Typically, there is fast initial recovery as the smaller components redistribute,
followed by a slower recovery phase as the mobility of larger species dominates.
It is difficult to resolve components if the difference in diffusion coefficients is
not large.
4. Notes
1. Argon-ion lasers are preferable to krypton-argon for this technique as they have
superior lifetimes, higher beam power, and lower maintenance costs.
2. Dialysis sacs should be double sealed for aggrecan and hyaluronan solutions as
they generate high osmotic pressures when dialyzed against water.
3. Allowing CNBr activation of carbohydrate chains to proceed for longer than
5 min results in depolymerization.
4.
The fluorescence concentration in Hi-Trap fractions can be checked quickly using
a standard UV gel transilluminator.
5.
Higher scan rates with larger pixel dimensions are needed to monitor the recov-
ery of very mobile species, therefore, a compromise is needed between increased
temporal resolution and decreased spatial resolution.
6
.
The gain and aperture of the photomultiplier tube must be set to zero during
bleaching to prevent damage to the tube.
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