 QuantaMetrics Inc. is in the unique position of providing the
necessary perfusion apparatus, and/or the necessary fluorescence instrumentation to do
this entire experiment. QuantaMetrics is a regional representative of the quantitative
ratio fluorescence systems available from Photon technology International Inc.
(PTI) in
the Midwestern United States and in Canada. QuantaMetrics is also the exclusive
distributor of the complete line of biomedical instruments available from Experimetria
Ltd. including their line of whole heart perfusion systems.
PTI offers instrumentation configured to perform the
fluorescence measurements and acquire multiple datasets of [Ca++]i,
pH, [Na++], and other ions quantitatively along with contraction, force,
LVP,
temperature and more from the perfusion apparatus. No modifications are required of the
Experimetria whole heart systems for simultaneous quantitative ion studies using the ratio
fluorescence technique. A PTI RatioMaster™ fluorescence system requires the purchase
of the special trifurcated remote fiber optic bundle (PTI Part Number 060-8012) as well as
connectors specific to the various PTI illuminator/detector alternatives. Link to PTI RatioMaster Systems. Link
to Experimetria Perfused Heart systems. Below is a general schematic of the apparatus. |
Sample Experimental Data:
For the experiment described here, retrograde coronary perfusion (Langendorff) with
constant coronary perfusion pressure was established with a supply of oxygenated
physiological saline circulated using a peristaltic pump. The isolated rabbit heart used
in this example was loaded by perfusing with 5 µM Fura-2 AM after careful preparation to
avoid circulatory problems. Hydrolysis of the indicator is critical and was assessed by
taking the fluorescence excitation spectrum of the intracellularly trapped dye (a
capability unique to PTI's system). Alternating UV excitation light of 340nm and 380nm
wavelengths with 3 nm bandpass was subsequently delivered to the sample, with the
fluorescence emission of intracellular Fura-2 detected at 510 nm. Intracellular calcium
transients and changes in the contractile force in the Langendorff-perfused beating heart
were measured to study the effects of various Ca++ channel blockers,
vasodilators, K+ channel openers, and catecholamines.
The measurements were performed by PTI in
collaboration with the Department of Physiology University Medical School, Debrecen
Hungary. The illuminator, a PTI high-speed multi-wavelength scanning system (shown at
right), delivers alternating wavelengths of light to the sample via a custom-designed
trifurcated quartz fiber optic bundle. The common end of the fiber optic bundle is
designed as a contact probe which delivers the excitation light to the sample. The probe
end is a 70mm long and 9mm diameter ferrule. The third leg of the bundle returns the
emission light to a PTI microscope photometer fitted with one or two PTI Model 810
photon-counting photomultiplier detectors. The fluorescence data is collected and analyzed
on a PC-compatible computer with PTI's proprietary FeliX™ Fluorescence Analysis
Software. This software also allows for the simultaneous acquisition and display of up to
6 other analog inputs such as contraction, LVP etc. Other hardware configurations are
available from PTI to accommodate different components and experimental requirements.