How to determine pixel size? Why do you include nano gold particles in your specimen?

[Hiroshi Imai]

Dear Carsten

Two questions in your tutorial are sitll in my mind over a month.

Could you please help me to better understand your tutorial?

1. How did you determine the pixel size (0.1062 nm/pixel)?

Could I use one of the layer lines of TMV for magnification calibration?

If you have any research articles (publications) under your recommendation regarding magnification calibration, could you please let me know?

2. Why did you include nano gold particles in your specimen?

What are the advantages to include nano gold particles in your TMV specimen?

In most of the single particle reconstruction, I do not see nano gold particles in ice.

Do you recommend the SPRING users to include nano gold particles if the users would like to repeat your experiment (by taking images of TMV using cryo-EM with a direct electron detector (e.g. Titan Krios EM equipped with Falcon II))?

Best regards,

Hiroshi

Hiroshi Imai (PhD)
 
Laboratory of Cell Motility,
Department of Biological Sciences, Graduate School of Science,
Osaka University,
Machikaneyama-cho 1-1, Toyonaka, Osaka, 560-0043, Japan
 

[Carsten Sachse]

Dear Hiroshi,

On Monday, 14 May 2018 12:21:49 UTC+2, Hiroshi Imai wrote:

Dear Carsten

Two questions in your tutorial are sitll in my mind over a month.

Could you please help me to better understand your tutorial?

1. How did you determine the pixel size (0.1062 nm/pixel)? 

Could I use one of the layer lines of TMV for magnification calibration?

We calibrated it using the known helical parameters from TMV. Yes, you can. As you can predict the positions of the layer lines (e.g. use SEGMENTLAYER2LATTICE or our web-based helical diffraction simulator at http://spring.embl.de), you can also calibrate your pixel size accordingly. Try to calibrate your images with the highest resolution layer lines that you still see.

If you have any research articles (publications) under your recommendation regarding magnification calibration, could you please let me know?

I am not aware of a publication that describes this procedure at a high level. Maybe other members of the discussion forum know this.

2. Why did you include nano gold particles in your specimen?

What are the advantages to include nano gold particles in your TMV specimen?

In most of the single particle reconstruction, I do not see nano gold particles in ice. 

Do you recommend the SPRING users to include nano gold particles if the users would like to repeat your experiment (by taking images of TMV using cryo-EM with a direct electron detector (e.g. Titan Krios EM equipped with Falcon II))?

The nanogold particles are not required for running Spring. You are totally right that commonly they are not included in a typical high-resolution single-particle type of imaging session. We wanted to use them to evaluate other data acquisition parameters. They are typically used in tomography experiments. 

Best wishes,

Carsten

[Hiroshi Imai]

Dear Carsten

Thank you very much for your clear reply.
It is fantastic to receive the answers from you.

I have started to repeat your experiment (data collection of TMV)
in order to better understand your high resolution cryo-EM image processing tu
torial.

Many thanks,
Hiroshi

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