A LIGHT-EMITTING DIODE MICROSPECTROPHOTOMETER - INTRACELLULAR CA2+ MEASUREMENTS IN ISOLATED STRETCH-RECEPTOR

A microspectrophotometer was designed to measure absorbance changes in single cells. The device utilizes sequentially activated light emitting diodes (LED) to provide different wave lengths of light. The instrument has the advantage of relative simplicity and less cost compared to other devices. The spectrophotometer was tested by measuring absorbance changes of the metallochromic Ca2+ indicator Arsenazo III (AIII) injected into the crayfish (Astacus astacus) stretch receptor. Under the conditions described the detection limit of the concentration of AIII was 0.05 mM and absorbance changes of 0.0005 can be reliably determined which correspond to a detection limit of 10-20 nM for free Ca2+ changes assuming a light path length of 0.003 cm and an apparent dissociation constant (K(D)) of 2 muM for the Ca2+-AIII complex. The upper frequency limit of the device is 3000 Hz. The absorbance measurements of AIII injected into the crayfish stretch receptor neurons revealed a Ca(i) of 375 +/- 177 nM (mean +/- SD: 14 cells). LiCl substituted for NaCl increased Ca(i) 45-100 nM in different cells, suggesting that a Na+ gradient is necessary for Ca2+ homeostasis in this receptor.