Biomedical Engineering Reference
In-Depth Information
6. Silicone grease provides a tight seal which will eliminate evaporation of the media for
many days, if the coverslip is initially well-bedded down. If desired, a small bead of
melted VALAP (1:1:1 mixture of vaseline, lanolin, and paraffin) can be applied around
the edges of the top coverslip to mechanically stabilize the seal (see Figure 3.3 ). We
use this extra sealing method when we need to change media during filming (see
protocol #8). Keep the amount of VALAP used to a minimum to reduce the chance that
any of the VALAP will deposit on the front element of the objective when the
preparation is in use. If this happens, use a small amount of Naphtha (lighter fluid) and
a cotton swab to clean the objectives.
7. Warm the assembled chamber to 37 C, place on the microscope stage, and start
filming.
8. Although these chambers support cell viability for more than 100 h, we often change
the media every few days to be safe. To do this, we stop the film run and take the
preparation off the microscope being careful not to disturb the
XY
position of the stage.
We sometimes mark one edge of the cell bearing coverslip with a felt tipped pen to
indicate the polarity of the preparation so that it can be replaced on the microscope
stage with proper orientation (see Figure 3.3 ). In the tissue culture hood, the bottom
coverslip (the one without cells) and surrounding area are cleaned with a cotton swab
soaked in ethanol. Sharp forceps are used to gently move the coverslip in both the
X
-axes to loosen the attachment (2 mm in each direction). Then, slide the
coverslip off and remove it prior to aspirating the medium and replacing it with fresh
warm buffered media (protocol #4). Ahead of time, flame a cleaned blank coverslip,
apply a thin rim of silicone grease around the edges of the coverslip, and expose it to
UV light for 10 min in a tissue culture hood. We then reassemble the preparation as
described in protocol #5, find the same field and restart the image sequence acquisition.
This procedure is also used to add or withdraw drugs from the cells. We use 6
- and the
Y
8
washes (total 5
6 ml of fresh media) to wash drugs out of the cells.
3.3 Correlative Live Cell
Fixed Cell Observations
Often studies require long-term time-lapse observation followed by fixation and
immunostaining of cells previously followed in vivo. The methodology described below has
enabled us to reliably mark and later relocate fixed cells for which we had acquired
long-term time-lapse sequences.
1. To mark the field of interest, we use a diamond scribe mounted in one of the positions
in the objective lens (e.g., Leitz Wetzlar, Germany) turret on the nosepiece of the
microscope. These scribes have the overall appearance of an objective lens with a
retractable diamond point instead of a glass front element. The scribe physically scores
a circle around the area of interest on the surface of coverslip. Lower the microscope
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