Biology Reference
In-Depth Information
Figure 7.4 Basic FLAP methodology using CFP and YFP-conjugated proteins. (A)
Cartoon depicting nuclear microinjection of plasmids encoding CFP- and YFP-conjugated
actin, which are expressed in the cytoplasm. (B) A collection of images of the same
fibroblast showing (i) CFP-actin localization, (ii) YFP-actin localization, (iii) phase
contrast, (iv) interference reflection microscopy (IRM). Note the co-localization of CFP-
and YFP-actin in stress fibres and how these terminate in the IRM dark patches that
represent focal adhesions. (C) Screen capture image from the Zeiss LSM 510 software
showing filters and mirror set-ups for collecting (i) CFP emission and phase contrast
images, and (ii) YFP emission and interference reflection images
After a suitable cell has been found, the image is rotated to fit into a
rectangular region of interest that defines the scan area. If a rectangular strip
is to be bleached across the cell, then the strip is defined horizontally in order
to reduce the number of scan lines in the bleach region and hence the
bleaching time. The gain for each channel is then adjusted so that the CFP and
YFP fluorescent intensities are matched without saturation and the offsets are
set so that backgrounds have zero intensity. The time-lapse mode is then used
to collect an image sequence. At least two frames are collected before
bleaching. Bleaching consists of 50 scans using the 514 nm laser line at
maximum power
for 2-3 s. Time-lapse
image
recording is
resumed
