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at the LDCS. In 3T3-L1 mature adipocytes, LD pairs are randomly selected
according to the close apposition of two LDs.
13. For selected LD pairs, it is important to eliminate the possibility that other LDs
are attached to the pair by careful visual inspection at different focal planes.
14. Select a circular bleaching area with 1-1.5
m diameter in the center of one LD
m
and start the photobleaching process.
14.2.1.2.4 Data preparation
15. Open the original image file in ImageJ and isolate the Bodipy 558/568 channel
through color splitting function.
16. Set the region of interest (ROI) to be a circular area at the center in the bleached
and unbleached LD, respectively. The diameter of the ROI is usually
one-third of the diameter of the LD. Perform the “Plugins/Intensity vs. Time”
analysis. This plugin could be found in MBF ImageJ for Microscopy
Collection ( http://rsbweb.nih.gov/ij/plugins/mbf-collection.html ) .
17. Export the intensity data to Excel and calculate the lipid exchange rate
according to the algorithm described below.
14.2.2 Data analysis and representative results
14.2.2.1 Algorithm for the calculation of lipid exchange rate
The rate of neutral lipid exchange is defined as the volume of neutral lipid exchanged
between two contacted LDs in a unit of time. The algorithm is developed based on
following assumptions:
1. Neutral lipids are constantly exchanged bidirectionally between two contacted
LD pair at the LDCS.
2. The thermodynamic properties of neutral lipids and the size of lipid-diffusion
pore(s) at the LDCS determine the neutral lipid exchange rate.
3. The duration of FRAP assay is short (within 5 min) so the change in LD size is
small and can be neglected during the assay.
4. Neutral lipids diffuse rapidly within one LD and intra-LD diffusion rate is higher
than that of inter-LD diffusion. Thus, the mean optical intensity reflects the
concentration of fluorescent-labeled neutral lipids (mainly TAG) of the measured
LD but not free fatty acids as Bodipy fatty acids are incorporated into TAG during
the incubation process ( Gong et al., 2011 ).
In our algorithm, the definition of each parameter is as follows:
'
, the neutral lipid exchange rate per LDCS; I , the mean optical intensity of the
measured area; V , the volume of total neutral lipids within one LD; and t , the duration
of diffusion process.
'
is a constant according to assumption (2).
V is a constant according to assumption (3) and was calculated by measuring LD
diameter in Carl Zeiss Zen.
We define a
V 2 , I 1, 0 and I 2, 0 are initial optical inten-
sities of LD1 and LD2. Thus, both a and b are measurable constants. By solving a
I 2 ; 0
I 1 ; 0
1
1
¼
V 1 þ
V 2 and b
¼
V 1 þ
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