Dear colleagues,
Our dataset was recorded with a Total exposure dose (e/A^2) of 100.
Since the total dose is very high, I wonder how to use/set 30 or 40 e/A^2?
At what stage can I do that?
Thanks in advance.
Kind regards,
Dmitry
which camera? since you recorded movies of frames, you can incorporate only the first some fraction of frames and remove the later ones.
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Hello @CryoEM2,
Falcon 4i, EER files.
Additionally, how to restrict the frames I want to incorporate?
Sincerely,
Dmitry
during the motion correction job, it asks how many frames to cut off from the front or from the back. I think start frame, end frame. not 100% how this works for EER converted to Tiff… you should also try it with the full exposure, as dose-weighting is also designed to help downweight the later, lower resolution frames. And obviously in the future collect only what you need, as time is money on a microscope - at 10e.p.s. we can get the whole dose in ~4s, many more movies per day.
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Thank you @CryoEM2 for the answer.
In my case, with 100 e/A^2 total exposure, the software takes care of overexposed images decreasing their impact on the signal – is that correct? And the control of the exposure – “dose-weighting" process occurs during the motion-corrected step? So, should I not change anything working with the full frame dataset? Could my resolution also get stuck due to the usage of these over-exposure frames (100 e/A^2), or is it already compensated during motion-corrected step?
I completely agree that for the time /efficiency reason, it is preferable to collect only the 30-40 e/A^2 frames. At the same time, some cases where the signal is quite low and the particles are barely visible may require to increase in the exposure time. (Of course, some additional filtering - “denoising” of the collected images, especially taken under low defocus, may help particle picking to some extent).
Kind regards,
Dmitry
yes, sounds like you’ve got it. in the old days they would take defocus pairs, where each high res image (with too little signal) would be accompanied by a second, high-defocus image so they could pick particles from the latter and apply to the former! genius! but slow.
I have no practical experience with your situation. yes, my guess is Dose-weighting would do a decent job and decrease the signal of the late frames so they wouldn’t negatively impact the resolution of the particles. But I would try 1) proceed as normal and 2) remove later frames and then have empirical evidence to guide future experiments. for funsies you can also 3) remove early frames keeping only those PAST 40é and verify that these are insufficient. In all cases, you can pick the particles using the high exposure and apply those coordinates to each set of micrographs during extraction - to have identical particle sets.
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thank you, @CryoEM2, for the useful suggestions.
As a future upgrade idea for the CS - it would be nice to have this option (dose weighting/exposure specification) during the Extraction step as well for the final refinement.
Kind regards,
Dmitry
CryoSPARC applies dose-weighting based on observations on a reference sample published by Grant & Grigorieff (2015). Increasing exposure should have no detrimental effect on the reconstruction, assuming the decay characteristics of your sample are sufficiently similar to those of the reference sample.
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Thank you for the suggestion. Dose-weighting requires movie input and therefore cannot readily be applied after motion correction.