Biomedical Engineering Reference
In-Depth Information
provide optical access to the brain, which can be done by using fi ne forceps, splints,
or a razor blade or an injection needle. An alternative approach is to completely
expose the brain and use this isolated brain preparation (Shang et al.
2011
).
Several types of imaging platforms such as a plastic cover slip or aluminum foil
have been used to mount the fl y (Fiala and Spall
2003
; Joesch et al.
2008
). An air-
supported ball system has also been used for imaging the brain of a behaving fl y
under tethered condition (Chiappe et al.
2010
; Kohatsu et al.
2011
). In all cases,
reducing movement artifacts by immobilizing the head is necessary to obtain high
signal-to-noise ratio. Beeswax, silicone wax, and dental glue that are harmless to
the animal are typically used to attach the fl y head to the imaging platform.
7.4.3
Preparation for Imaging Sensory Organs
Because sensory neurons of insects are often associated with specialized cuticular
structures such as bristles, hairs or sensillae, nondestructive imaging through the
intact cuticle is in these cases required. Such noninvasive imaging is possible for
sensory neurons located closely underneath the cuticle (Kamikouchi et al.
2009
,
2010
; Pelz et al.
2006
). A fl uorescent sensor protein with strong baseline fl uores-
cence is useful to locate the sensory neuron of interest before the onset of the stimu-
lus. Also in this case, immobilizing the body parts to be imaged is, of course, a
critical step to get high signal-to-noise ratio.
7.4.4
Equipments and Data Analysis
In general the imaging equipment includes a fl uorescence microscope, a light source
for fl uorophore excitation, and an emission light detector. Although the most power-
ful systems for Ca
2+
imaging are probably laser-scanning microscopes, e.g., confo-
cal laser-scanning or the two-photon microscopes, a simple combination of a
standard upright epifl uorescence microscope and a charge-coupled device (CCD)
camera can for most applications deliver high-quality images. Once time series
images of the GECI emission intensity are recorded, their fl uorescence intensity can
be analyzed off-line. Image processing, fi ltering, and averaging can all be used to
eliminate noise and enhance calcium signals. For details of the imaging equipment
and data analysis, see Part 3 (“Optical recording techniques”).
7.4.5
Control of the Expression Level
Because GECIs are also Ca
2+
-buffering proteins and therefore are potentially cyto-
toxic, optimizing the magnitude of expression to balance signal levels and
