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2. The FRAP Profiler plugin generates several windows ( Fig. 7C ):
i. A log file with the parameters of the curve fit (half-life, immobile frac-
tion, and constants). For a single exponential curve fit, the form of the
equation with constants is p[0]*(1-e (-p[1]*x).
ii. If the option was selected, a window containing an image showing the two
ROIs.
iii. Graphs of (i) the raw FRAP data; (ii) normalized FRAP data; and (iii) curve
fits of the normalized data, including curves with the first postbleach time
point set as the origin (i.e., t = 0). By clicking the ''List ... '' button, the data
in the latter can be exported to a spreadsheet or curve-fitting program for
further analysis.
D. Correlative Fluorescence and TEM During Morphogenesis
(see Sims and Hardin, 2007 for more details)
1. Introduction
This final section describes high-pressure freezing (HPF) techniques for correl-
ative light and electron microscopy on the same sample, starting with embryos
mounted for 4D microscopy. Fluorescence information from a whole mount can
be displayed as a color overlay on transmission electron microscopy (TEM) images
to generate what we have termed fluorescence-integrated TEM (F-TEM) images. An
alternative for postembedding correlative TEM is described elsewhere ( Sims and
Hardin, 2007 ). The method we describe here uses a thin two-part agarose pad to
immobilize live C. elegans embryos for LSCM, HPF, and TEM. Pre-embedding F-
TEM images display fluorescent information collected from a whole mount of live
embryos onto all thin sections collected from that sample. For typical uses in our
laboratory, this method relies on creating a strain with a rescuing array containing a
GFP tagged protein of interest, often rescuing a lethal mutation (e.g., ajm-1;
Koppen et al., 2001 ). Embryos that have lost the rescuing transgene arrest.
Alternatively, transgenes can be used as simple markers to identify the genotype
of embryos. Because C. elegans embryos are difficult to fix with conventional
chemical fixation, HPF is the method of choice. The agarose mount we describe
for embryos is used for ultrastructural analysis, using freeze substitution with 1%
osmium and 0.1% uranyl acetate. The agarose pad is made of a thin base of high-
strength agarose. The thin base layer provides the strength and toughness to keep the
mount intact. An agarose pad composed only of low-melting agarose would not hold
the pad together well enough to allow transfer to a HPF specimen carrier.
Additionally, it is necessary for the top agarose layer to be very thin to allow imaging
in a confocal microscope. The method described here is specifically designed to be
used at the conclusion of a 4D experiment. An alternative correlative procedure has
been described elsewhere ( Kolotuev et al., 2010 ).
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