FLUO-3 is a fluorescent dye that can be used to measure the concentration of free Ca2+\text{Ca}^{2+}Ca2+ ions inside cells.
FLUO-3 contains four negatively charged carboxylate groups (COO−\text{COO}^-COO−) and has a relatively large molecular mass of approximately 570 g mol−1570\text{ g mol}^{-1}570 g mol−1. Use this information to explain why FLUO-3 is unable to cross cell membranes without chemical modification.
Scientists used FLUO-3 to study the role of Ca2+\text{Ca}^{2+}Ca2+ ions in synaptic transmission between a motor neurone and a postsynaptic muscle fiber. FLUO-3 was injected directly into the synaptic bulb of the motor neurone, and its fluorescence was observed using a laser scanning confocal microscope. (i) Explain one advantage of using a confocal microscope rather than a standard widefield light microscope in this study.
The motor neurone was stimulated electrically at three different frequencies: Low, Medium, and High. At a Low stimulation frequency, the FLUO-3 fluorescence inside the synaptic bulb was Low and the peak membrane potential of the postsynaptic muscle fiber was −70 mV-70\text{ mV}−70 mV. At a Medium stimulation frequency, the fluorescence was Medium and the peak potential was +30 mV+30\text{ mV}+30 mV. At a High stimulation frequency, the fluorescence was High and the peak potential was +30 mV+30\text{ mV}+30 mV. The intensity of FLUO-3 fluorescence is directly proportional to the concentration of free Ca2+\text{Ca}^{2+}Ca2+ ions. The scientists concluded that an increase in the concentration of Ca2+\text{Ca}^{2+}Ca2+ ions in the presynaptic neurone causes an action potential in the postsynaptic cell. Evaluate and explain the scientists' conclusion.
