Intracellular metabolic state is one of the main determinants of regulatory adjustments. A number of in vitro techniques were developed to assess variables (NADH/NAD+, redox state, intracellular free calcium) closely related to this state. Quantitative, in vivo evaluation of these variables, however, was difficult to perform, primarily due to the presence of a significant, but hard to estimate "hemodynamic artifact" - i.e. the non-specific changes caused by absorbing/scattering effects of the fluctuating blood content in the tissue under investigation - in the optical signals. A quantitative, multiwavelength fluorescence imaging technique was developed to minimize the effect of the hemodynamic artifact during in vivo measurements. The technique is based on the use of a sensitive cooled slow scan digital CCD camera attached to an intravital microscope and enables the selection of practically avascular tissue microregions of interest (>10 ìm x 10 ìm). The new technique was tested in both the trans- (rat spinotrapezius and uterine muscles) and epi-illumination (rat EDL and soleus muscles) mode. Either single wavelength or ratiometric measurements could be performed. The imaging technique enables corrections for motional artifacts and limited tissue density changes. Although the technique does not directly provide absolute values for the variables evaluated, with proper in vitro calibration processes quantitative estimate of intracellular concentration changes can be obtained.
|Publication status||Published - Mar 20 1998|
ASJC Scopus subject areas
- Molecular Biology