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Tutorial: Negative Stain Data

Learn how to work with negative stain data in CryoSPARC.

How to specify negative stain data

You can specify the data is negative stain, at the time of import, or manually.

Import negative stain movies or micrographs

When importing negative stain movies or micrographs, use the Negative Stain Data toggle in the Import Movies or Import Micrographs jobs to indicate that you are processing negative stain data (light-on-dark). Subsequent jobs that use the imported negative stain data, will adjust their relevant parameters accordingly (for more details, see below: Jobs affected by negative stain data).

Import negative stain particles

If importing particles, specify the type of data being imported in the Import Particles job. Subsequent jobs that use the imported negative stain data, will adjust their relevant parameters accordingly (for more details, see below: Jobs affected by negative stain data).

Manually set negative stain values for an exposure dataset

Use the Exposure Tools job to manually set the Negative Stain and Phase Plate Data values for an exposure dataset:

Turn off CTF correction

For negative stain data, CTF correction may decrease the quality of 2D classification and reconstruction results. To bypass CTF estimation, the data can be imported in the Import Movies or Import Micrographs job with the Output Constant CTF toggle on:
Alternatively, the 2D Classification job can be run without CTF correction, by setting the Do CTF correction toggle:

Jobs and parameters affected by negative stain data

  1. 1.
    Import Movies: "Negative Stain" toggle will set mscope_params/neg_stain field to 1 (True)
  2. 2.
    Import Micrographs: "Negative Stain" toggle will set mscope_params/neg_stain field to 1 (True)
  3. 3.
    Import Particles: "Data Sign" drop-down list will set blob/sign field to +1 (cryo-EM data) if dark-on-light is selected or -1 (negative stain data) if light-on-dark is selected. The default is light-on-dark, as cryo-EM data is usually inverted during processing.
  4. 4.
    Exposure Tools: Allows user to set mscope_params/neg_stain and mscope_params/phase_plate manually.
  5. 5.
    Manual Picker: Extracted particles are indicated with the correct sign (+1 for dark-on-light particles and -1 for light-on-dark particles)
  6. 6.
    Template Picker: Templates used to pick particles are flipped (dark-on-light to light-on-dark)
  7. 7.
    Blob Picker: Blob templates used to pick particles are flipped (dark-on-light to light-on-dark)
  8. 8.
    Local Motion: Extracted particles are indicated with the correct sign (+1 for dark-on-light particles and -1 for light-on-dark particles)
  9. 9.
    Extract From Micrographs: Extracted particles are indicated with the correct sign (+1 for dark-on-light particles and -1 for light-on-dark particles)
  10. 10.
    Topaz Train: Preprocessed micrographs are inverted (light-on-dark to dark-on-light) before being used for model training
  11. 11.
    Topaz Extract: Input micrographs are inverted (light-on-dark to dark-on-light) before being processed by Topaz Extract
NB. Certain jobs plot particles while taking into account their particle sign (+1 for dark-on-light (cryo-EM data) and -1 for light-on-dark (negative stain data)). You might notice that your negative stain data looks like cryo-EM data in the plot, but please note that the actual particles on disk are still negative stain data.

Data type reference chart

Data Type
Data Sign
Description
Cryo-EM
+1
dark-on-light
Negative Stain
-1
light-on-dark
Note: Cryo-EM data is typically recorded as +1 (dark-on-light) but is often inverted during processing.
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