OGB-2 is a fluorescent indicator dye that can be used to measure the concentration of free Ca2+\text{Ca}^{2+}Ca2+ ions inside active neurones.
OGB-2 contains five negatively charged carboxylate groups (COO−\text{COO}^-COO−) and has a relatively large molecular mass of approximately 1120 g mol−11120\text{ g mol}^{-1}1120 g mol−1. Use this information to explain why OGB-2 is unable to cross cell membranes without chemical modification.
Scientists used OGB-2 to study the role of Ca2+\text{Ca}^{2+}Ca2+ ions in synaptic transmission between a nociceptive sensory neurone and a postsynaptic spinal interneurone. OGB-2 was injected directly into the synaptic bulb of the sensory neurone, and its fluorescence was observed using a laser scanning confocal microscope.
Explain one advantage of using a confocal microscope rather than a standard widefield fluorescence microscope in this study.
The sensory neurone was stimulated electrically at three different frequencies: Low, Medium, and High. At a Low stimulation frequency, the OGB-2 fluorescence inside the synaptic bulb was Low and the peak membrane potential of the postsynaptic spinal interneurone was −60 mV-60\text{ mV}−60 mV. At a Medium stimulation frequency, the fluorescence was Medium and the peak potential was +35 mV+35\text{ mV}+35 mV. At a High stimulation frequency, the fluorescence was High and the peak potential was +35 mV+35\text{ mV}+35 mV.
The intensity of OGB-2 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.
