And again, in 1980s, people started using optical masses to demonstrate or prove during a, Depolarization in a prisoner terminal. There will be indeed calcium influx into left terminal. And this is one of the experiments using the AR3 calcium sensitive dye, okay? And again, in the screen of terminal, you can see that if you're depolarizing the presynaptic membrane to potential. And you can see that association of the calcium influx reporting by this AR3 indicators, okay. This AR3 indicator is not so good because they are based on the absorbance change. So it's not so sensitive, it's not frozen changes. So it's like a phenome, phenalref for example, they only change their absorbance, their color. But it's not a first sense, so now people have designed better indicators. And then Roger Chen, from UC San Diego, when he was an assistant professor at Berkeley, he designed a series of calcium indicators that, based on EGTA and BAPTA, okay, where he link this BAFTA-like curator, which you can specifically bind into calcium with different chromophore or fluo-4. And using this organic compound, he demonstrated you can generate a calcium synthesis. That upon calcium binding, that you would have first in signal changes. So for example, this is one of the calcium dye fluo-3, okay? And you can see that upon the different calcium concentration, it will emit different forces. And this will be much more sensitive than the AR3 indicators, which is purely based on absorbance change because you can use the exact excitation light and only collect specific wavelengths of emission light. And you can measure in a calcium dependent way that force in sigma change. Why calcium binding will change its forces? Well, if you look at this structure of this molecule, you can see that when calcium binds to it, it will alter the electron in this nitrogen ion, okay. Because this nitrogen ion originally was pi-pi conjugate with this fluo-4, okay, so the calcium binding will distraught the pi-pi electron covering to this fluo-4 and will change its quantum yield. Okay, and therefore, the frozen signals will change. So, it's very sensitive to calcium because of magnesium or other ions. We will not bind to this EGTA or BAFTA-like platform at the top, okay? Indeed, you can make it even more derivative of this cut indicator, and this is also another very useful one, one of the first, called radiometric calcium indicator, Fura-2. Again, you use the similar platform of the BAFTA here. But the conjugated upon calcium binding, it has this unique property, excitation spectrum will change their shape, okay. Let's just look at it in detail, okay. So with the different level of calcium, the excitation spectrum will change the shape. For example, this 340 peak, when calcium level goes down, it will go down. We are to the 380 level peak, will corresponding to increase, okay. And in between these two peaks, there's a wavelength. At this point, this is a so-called isopoint that which is insensitive to calcium concentration, okay. So this indicator actually is very useful. The reason is that with different calcium level, it changed its shape, okay? So the shape, or more specifically, you can just take the ratio of these two peaks, is calcium sensitive, rather than the pure intensities calcium sensitive. Why this is useful? The reason is that if it's only intensity rather than the shape, it's calcium sensitive, the main sense will affect the intensity. For example, if a cell is very big, it's very thick, okay. With a thin concentration of the indicators, big fat cells will be having much larger emitting intensity than a much thinner cell, right? On the other hand, if you have a much more sensitive camera in your lab, than your neighboring lab, okay? With the thin concentration of the indicator and thin concentration of calcium, with more sensitive cameras, you will get higher intensity, okay? So the intensity alone is not so quantitative. You will be associated with both of the strings of excitation light, the thickness of the sample, the concentration of the indicator, the sensitivity of the detector, okay? And that will make the comparisons between the experiments very, very difficult to do, okay. But if we are using the shape or the ratio of these two peaks, high peak and going down peak, then largely the ratio will be insensitive to a lot of factors that I mentioned, okay. It doesn't matter how strong your excitation light is, because as long as you are using the same excitation light, the ratio between those excitation peaks will normalize, will cancelling out, the intensity, okay, the excitation strength. So this has been very useful, okay. And again, using that, people can achieve much more sensitive detection of calcium signals.
