Radiometric Fluorometer for Tissue Ion Measurements

Within the last six years, ratiometric fluorescent ion indicators such as fura-2 and indo-1 (for calcium), SNARF-1 and BCECF (for pH) together with newer agents to measure sodium, magnesium and potassium have become widely accepted as useful tools to monitor the intracellular environment. More recently, photolabile chelators have been used to control the release or sequestration of cellular modulators such as calcium, cAMP and ATP. Although much of this work has been performed on isolated single cell preparations, there are numerous questions that can be more effectively or conveniently answered by experiments using multicellular preparations, such as tissue strips, rings or isolated organs. However, a major limitation is the lack of equipment designed specifically to make both fluorescence and mechanical measurements on these preparations. This is surprising since multicellular preparations have been a mainstay in physiological and pharmacological research for decades. To address this need, a ratiometric tissue fluorometer will be constructed to allow utilization of many of the currently available ratiometric and non-ratiometric fluorescent probes, alone and in combination. The system will be instrumented to permit simultaneous measurements of fluorescence of one for more indicators and developed force or perfusion pressure/flow, together with control of preload, addition of drugs and bath wash cycles. The fluorometer will be demonstrated in both smooth and cardiac muscle preparations, which have temporal cation responses that span the range of multicellular preparations.