Hardware requirements

Bayesian analysis of blinking and bleaching

Hardware requirements

Postby docfra » Sun Feb 17, 2013 6:20 pm

Hi list,
I have a question regarding microscope hardware requirements. In particular I would like to know:
1. if TIRF illumination is a requirement for 3B microscopy
2. if upright scope can be used for 3B microscopy

Thanks,
Francesco
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Re: Hardware requirements

Postby susancox » Mon Feb 18, 2013 2:06 pm

Hi Francesco,

1) TIRF illumination is not a requirement for 3B microscopy, in fact the original Nature Methods paper did not use TIRF. However, if you are using standard widefield microscopy you generally to be looking at a sample where the labelled parts are either thin or sparse or both. This is because if you have too much out-of-focus light then the background is very high and it will not be possible to do a good fit to blinking events.

This is an issue for all methods which try to fit bleaching/blinking events - the higher the background, the worse the resolution of the reconstructed image.

2) An upright scope could be used, provided it had a powerful enough illumination source to allow the observation of blinking at a useful timescale. So if you have a good quality arc lamp on it, it should be fine. If you are doing experiments in live cells you have to balance how fast the features you are interested in are moving, so if you wanted to observe something that was moving fast you would need a laser source to avoid motion blur.

Regards,
Susan
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Re: Hardware requirements

Postby docfra » Fri Mar 28, 2014 6:34 pm

Dear Susan,
thanks for the information and I apologize for the delay in replying to this. I moved to a different lab and for a complete set of different reasons, I came back to 3B microscopy.

I still have a technical question though: do I need an emCCD camera even for fixed samples? or is that a requirement for live imaging of fluorescent proteins?

Thanks
Francesco
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Re: Hardware requirements

Postby susancox » Wed Apr 02, 2014 9:08 pm

EMCCD cameras generally have a better S/N ratio and sensitivity and so would be my first choice.

It is possible to do localisation with standard CCDs, but you need more photons per molecule to achieve the same quality of reconstructed image. So you will pretty much always be better off with EMCCD. I haven't tried getting 3B to work with a CCD, it would depend on the brightness of the sample and the blinking properties as to whether you could get good enough data.

If you mean SCMOS, then it's a more complex issue. Firstly, you'd need to make absolutely sure that you use it in a mode where hot pixels are highlighted. Many SCMOS come with a mode, often by default, where the hot pixels are replaced by an average value from surrounding pixels. This will throw localisation positions off by a long way. There is also the issue that the noise from an SCMOS camera is different to the noise from an EMCCD camera, so the noise model should be changed. We have not implemented this in 3B yet. I have, however, imaged a known structure with 3B using an SCMOS without changing the noise model, and the image looked similar to 3B with an EMCCD, and to images obtained with STED. So the incorrect noise model does not seem to produce artefacts. I suspect the inaccuracies produced by the noise level being wrong are small on the scale we are dealing with, since 3B is lower resolution than other localisation techniques. If you want to know more about issues involved in using SCMOS cameras, Huang and Bewersdorf published a good paper on it in Nature Methods last year.
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Re: Hardware requirements

Postby docfra » Wed Apr 02, 2014 10:28 pm

Thanks for the very useful information.
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Re: Hardware requirements

Postby nawaz » Mon Jan 05, 2015 11:52 am

this is very nice post
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