Biology Reference
In-Depth Information
photobleaching of less stable fluorochromes. Once the field is selected, images of the
more easily bleached fluorochrome should be collected first—in this case the BOD-
IPY lipophilic dye. Once a field is chosen, it is usually best to use an expose time that
saturates a few pixels in the brightest area. Larger regions of saturation may be nec-
essary; for example, in
Fig. 12.1
(ER-marker panel), clear depiction of the “lacy” ER
at the cell periphery required exposure times that resulted in a large area of saturation
in the perinuclear region. To identify representative data, image multiple fields.
When assembling a figure comparing multiple conditions, choose fields where the
cells are similar in size and shape.
Humans perceive green structures as sharper and brighter than red structures; for
this reason we suggest presenting the individual channels as grayscale images. Merged
color images are more easily interrupted when the most sharply defined structure is
depicted in green. For example, the perilipin 1 signal in
Fig. 12.3
sharply outlines
the more nebulous BODIPY 493/503-labeled neutral fat. This image is more under-
standable when the perilipin 1 signal is shown as green and the BODIPY 493/503 sig-
nal as red. Note that this presentation reverses the wavelengths at which the separate
channel images were collected, and hence requires explanation in the text of reports or
manuscripts. Finally, to assure images are published at a size large enough to show the
relevant features, use a font size that is only legible when the relevant features are ap-
parent. It is also helpful to submit images in a size that is compatible with journal spec-
ifications to avoid arbitrary resizing by the typesetter.
SUMMARY
Proteins forming a coat around intracellular fat droplets have been identified, and
antibodies against fat droplet proteins can be conveniently imaged using standard
epifluorescence microscopy. In addition, the lipid core of fat droplets in living
and fixed cells can be directly labeled by lipophilic incorporation of BODIPY dyes,
or in living cells by the metabolic incorporation of BODIPY-conjugated fatty acids.
In this way both lipid cores and the surrounding protein coats of fat droplets can be
imaged simultaneously. Placing fat droplets in subcellular context, especially
with respect to the ER and mitochondria, is also possible with these methods.
Cross-linking fixation is superior because methanol fixation disrupts fat droplet mor-
phology. Fat droplets are most conveniently imaged in cells grown directly on glass
coverslips in individual wells of tissue culture plates. This format makes multiple
experimental conditions feasible and analysis by imaging practical.
Acknowledgments
This work is supported in whole or in part by the National Institutes of Health Grant R01
DK088206-01A1 (N. E. W.). Postgraduate support (to J. R. S.) was from the Training Program
