Does anyone have experience or tips using Andor iXon EMCCD in the “fast kinetics” mode? From what I understand, the fast kinetics mode allows the camera to image with microsecond time resolution.
I am trying to use TIRF to get on/off rates for proteins binding. There is a nice example I found in the literature with a similar system, but I am not sure where to start to set up this system or how straightforward this is to implement. (link to paper: https://www.molbiolcell.org/doi/full/10.1091/mbc.E14-11-1539)
Any resources or suggestions would be much appreciated.
Yes, we are using elements. I looked around on the acquisition settings, but I didn’t see anything obvious (at least to a novice like me), so I figured I’d check in here.
I’m not 100% sure, I’ve never used that mode. But I can give a couple quick tips, and maybe we’ll find someone who has specifically used that mode.
In elements, in the camera widget (where you control exposure and stuff), there is a dropdown menu towards the bottom of the widget called “advanced” or something like that. Exactly what you find in there will depend on the camera… but for iXon camera’s you’ll likely find stuff related to these modes in there. On thing I have used, and I know you’ll find in there, is something called “Vertical Pixel Shift Speed” (or something like that), along with “Vertical Clock Voltage Amplitude” (or similar). These are related to andor’s fast readout mode, but not necessarily the full blown fast kinetics mode. If you reduce the pixel shift speed to the lowest value, you’ll likely get some nonsense at first, and will need to bump up the clock voltage slightly. That can help achieve higher frame rates, assuming you’ve set it to the highest EM readout speed available, and are not limited by exposure times.
That said, I think that Andor’s fast-kinetics mode is technically/actually a more elaborate mode than simply increasing vertical shift speed. I’m not exactly sure how/whether elements integrates support for it, or how it will present itself in the advanced options widget in elements (but have a look). One other important thing to note from the Andor document I linked above: that mode does require physically preventing light from hitting anything but the specific sub-array you’ve selected. So, you’ll need some sort of an aperture to truly use that mode (i.e., it’s not strictly a software feature, it requires some degree of hardware accommodation). Perhaps others here will have more direct experience with that mode
an update @jk7039, we’re at QI now and I had a chance to look at an iXon camera on elements. I think what you’re looking for is referred to as “isolated crop mode” in elements.
So, you’d click on Commands -> Advanced Camera Settings...
I can’t tell you for sure whether isolated crop mode is the same as fast kinetics mode… I would suggest you reach out your andor rep for clarification on that
yeah, I agree that it sounds like the fast kinetics mode. I’m gonna do some pilot experiments next week, so I’ll try this out then.
Regarding the design of the experiments, I assume I will have to make a photobleaching correction. I am also thinking about how to optimize the acquisition parameters so I can get the highest signal possible with sufficient temporal resolution for monitoring the kinetics.
Do you have any thoughts on differences between “isolated crop mode” vs “triggered acquisition” regarding how to figure out the max theoretical temporal resolution? Or is isolated crop mode using triggered acquisition?
(not sure if that makes sense)
I have been working through the camera user manual to better understand… but maybe I need to come back to QI lol
