Hello! I am just starting to explore self-labeling tags technologies in the course of my internship in MPIMF Heidelberg, and am playing around with the machines that are available. One is Tecan Spark 20m, and I'm trying to make sense/optimize the usage of it for tag binding efficiency evaluation and potentially other related processes.
I was wondering, what exactly do Gain and RFU parameters do with the signal? I understand that Gain is essentially an amplification of the signal once it's been transformed from light into electricity. But what is the point of RFU? Isn't it somewhat just the opposite of Gain (well, in different units), so what is the actual point of having those two parameters next to each other? Wouldn't it be sufficient to have just one Gain for example? And how do you work with it in your lab?
Any input is highly appreciated, thank you!
Artur
Hello, John! I am referring not to the RFU units, which indeed appear in fluorescence intensity measurements, but to the RFU parameter (yes, it's a little confusing) that is next to Gain parameter in the Fluorescence Polarization setup screen, it is also expressed in percentage.
Since you can go between 0-100% there, it seems to me that it limits the Gain of signal in that way, but I fail to see how it is useful since you can just manually reduce gain, so I was asking if anyone experienced with the machine could advice me on that.
Hi Artur, I spend some years of my life as a system engineer developing the Tecan Spark series. Pulling nights and weekends, Donating blood, sweat and tears (well, not tears really), creating inventions for 6 new patent applications, and finally get laid off after the product being launched. Tecan readers might be great products, but as an employer, the company utterly sucks...
About your question:
1) it is explained in your manual, just read it!
2) Tecan support has to answer all user questions. Give them a call!
3) it means that you can set the auto-gain to meet a percentage of the intensity scale (RFU), so that if the signal increases later on, you will not run into saturation. This is good for time kinetic cycles, when you expect the signal to increase over time, but still need to decide for a gain value at the beginning. You don't want to have the gain adapted during the kinetics measurement, because there is a non-linear relationship between gain and fluorescence signal.
Now I've done the job for Tecan again, and don't even get paid for it...
Hello, Friedrich!
I wouldn't have gone to post here before reading the manual, but sadly it doesn't provide comprehensible information about the RFU scale. Thank you for your answer, I think I figured it now
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