the highest available pressure applied, position the stage such
that the tip only slightly reaches trough the edge of the glass
slide or coverslip. Slowly lower the needle until the tip bends
and snaps onto the other side of the glass. Now, slowly raise the
tip till it bends and snaps back to focus. Usually, this maneuver
opens the tip without affecting the final diameter and thus the
piercing ability. However, if it fails repeatedly or the tip gets
broken, replace the needle and start over fromstep 3of Sub-
heading3.7.- Exposing the tip to open air for prolonged periods will cause
clogging, so keep it under the oil also when preparing or
imaging the specimen. - Assessing the axial position of the tip relative to the specimen is
difficult. As a rule of thumb look for indications of physical
interaction, e.g., movement, rotation and distortion of the
specimen and bending of the tip. Movement of the specimen
indicates too large tip diameter and a consequent inappropriate
piercing pressure, whereas rotation of the egg chambers sig-
nifies too lateral hit which usually stems from a too shallow
angle of the needle. Ideally, this angle is between 30 and 45
relative to the glass plane holding the specimen. - It may happen that the tip gets stuck in the perivitelline space
between the oocyte and the follicular epithelium. In such cases,
formation of a liquid bubble can be observed after injection,
accompanied by an immensely bright fluorescent signal of the
probe around and within the oocyte due that it gets endocy-
tosed by the oocyte into yolk granules. Due to the intensity of
this aspecific fluorescence, the affected oocyte cannot be used
for mRNP imaging even after reinjection. - TO and QB labeled probes can be simply combined with simul-
taneous imaging of red (e.g., mCherry) or green fluorescent
proteins (e.g., EGFP), respectively due to the differences in
the emission and absorption spectra of these fluorescent mole-
cules. Using microscopes equipped with a supercontinuum
based light source (e.g., a white-light laser) TO can be combined
with EGFP with minimal cross-talk using the following sequen-
tial scan setup: 470 nm laser line and 480–520 nm detection
window to image GFP and 525 nm laser line and 535–575 nm
detection window to image TO (Fig.2). - We found no decline inoskarmRNP motility within the first
hour after dissection. Very little or no RNP motility and a
subsequent formation of larger RNP aggregates over the
course of an hour are indicative of either inefficient insulin
signaling or probable interference of the probe with normal
mRNP biogenesis. To distinguish between the two scenarios,
repeat the microinjection and imaging using cultivation
medium made with a fresh aliquot of human insulin.
286 Jasmine Chamiolo et al.