24
World Precision Instruments
that have had their membranes removed or made permeable. The removal of
membranes from the muscle fi bers permits the free movement of molecules
between the cells and their incubation solutions.
During the course of the experiment, the skinned muscle fi ber preparation is
incubated in solutions that contain the enzymes and substrates needed for the
reactions of the muscle contraction. Each solution in the series causes the skinned
fi bers to contract to a varying degree. While the tension of the fi bers is being
recorded, the fi bers are illuminated with a beam of UV light to fl uoresce the NADH in
the incubation solution. As ATPase activity occurs, the fl uorescence of the incubation
solution that is measured by the fl uorometer decreases. Since the decrease in NADH
fl uorescence is proportional to the increase in ATPase activity, the slope of the
fl uorescence decay curve provides a measure of the rates of ATPase activity and
ATP consumption in the fi ber in proportion to the amount of force that the fi ber
generates.
Although ATPase cannot be measured directly, the correlation between ATPase
activity and the conversion of NADH to NAD allows ATPase activity to be measured
by the change in the fl uorescence of the incubation solutions containing NADH.
NADH is auto-fl uorescent and has a maximum absorbance at 338nm with an
emission maximum at 507nm.
NOTE
: When performing a single-emission, single-excitation measurement with
NADH, the proximity of the fi ber-optic probe to the prepared tissue has a signifi cant
infl uence on the intensity of fl uorescence that is collected. If the probe is adjacent
to contracting tissue, movement artifacts can cause fl uctuations in the amplitude of
the fl uorescence being recorded. This problem is eliminated through the use of a
secondary fl uorophore to track the motion artifacts in the fl uorescence recordings.
In this case, a convenient secondary fl uorophore is TAMRA. Although the maximum
absorbance occurs at 552nm, TAMRA has a secondary absorbance peak at 355nm,
which is adjacent to the 360nm light used to excite NADH. The emission maximum
of TAMRA occurs at 585nm.
Percent Signal
0
20
40
60
80
100
Wavelength [nm]
300
400
500
600
700
360nm LED [emission]
NADH [excitation]
NADH [emission]
TAMRA [excitation]
TAMRA [emission]
472nm Filter [transmission]
572nm Filter [transmission]
Fig. 42— Excitation, emission, and fi ltering of NADH and TAMRA with the BF-100
Biofl uorometer.
