Biomedical Engineering Reference
In-Depth Information
UTRs; for tagging
cdc3
(um10503) mRNA,
boxB
hairpins were inserted 100 nt
downstream of the stop codon.
19. At fi rst, P
crg1
is induced because switching on P
nar1
and P
crg1
promoters simultaneously results in a delay of fi lament
induction.
20. Due to the relatively high cytoplasmic signal intensity of
ʻ
[
35
] help to determine the length of 3
′
N
*
G
2
, we recommend to draw the line for the kymograph
point by point to ensure the selection of the pixel with the
highest intensity for the kymograph line. The thickness of the
kymograph line should not exceed three pixels.
21. This setup is optimized for comparably slow fl uorescence
recovery processes in the order of minutes. For recovery in the
order of seconds, settings have to be adapted for faster
acquisition.
22. Before acquiring Gfp fl uorescence with the LED, the software-
driven autofocus was used in the DIC channel to assure that
acquisition started in the correct focal plane. The whole proce-
dure can be automated with the “multidimensional acquisi-
tion” tool of MetaMorph 7.
23. In our experiment, we determined fl uorescence recovery of a
cortical protein. Acquiring
z
-stacks might not be required if
studying cytoplasmic proteins and are not applicable if recov-
ery processes in the range of seconds are analyzed.
24. Shrinking of the agarose cushion requires drift correction.
25. Identify a background region of a certain size that can be used
in all experiments.
26. With every
z
-stack taken, the Gfp fl uorescence gets photo-
bleached to a certain amount. One has to correct for this
acquisition bleaching. Also correct for collateral bleaching.
The high intensity of the 405 nm laser causes bleaching in
areas outside the determined area.
27. A small population of mEosFP emits red fl uorescence already
before irradiation with 405 nm light. That is an original char-
acter of mEosFP.
28. As the pool of non-converted mEosFP is depleted during data
acquisition, it is advisable to increase the power of the activa-
tion laser to keep the number of activated fl uorophores per
frame constant.
29. For a typical image size of 256 × 256 pixels per image in 16-bit
tif format, the processing time is 15-30 ms per image, depend-
ing on the complexity of constructed image. The PALM
micrograph in Fig.
6a
is constructed by 1,000 frames with a
time resolution of 50 ms.
