Tutorial: Negative Stain and Phase Plate Data Workflows in cryoSPARC

Last Updated: January 28, 2020

In cryoSPARC v2.13+, processing of negative stain and phase plate data are seamlessly supported. Using a simple toggle, users can indicate whether the exposures they are importing are dark-on-light (cryo-EM data, +1) or light-on-dark (negative stain data, -1). Users can also specify phase plate data when importing by turning on the "Phase Plate Data" toggle in Import Movies and Import Micrographs.

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.

Negative Stain Workflow

Negative Stain Phase Plate 1

Use the Negative Stain Data toggle in the Import Movies and Import Micrographs jobs to indicate that you are processing negative stain data (light-on-dark). This will ensure particle picking and reconstruction work properly later on in your workflow.

Negative Stain Phase Plate 2

Specify the type of data being imported in the Import Particles job.

Negative Stain Phase Plate 3

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

Jobs Affected by Negative Stain Data

  1. Import Movies: "Negative Stain" toggle will set mscope_params/neg_stain field to 1 (True)

  2. Import Micrographs: "Negative Stain" toggle will set mscope_params/neg_stain field to 1 (True)

  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. Exposure Tools: Allows user to set mscope_params/neg_stain and mscope_params/phase_plate manually.

  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. Template Picker: Templates used to pick particles are flipped (dark-on-light to light-on-dark)

  7. Blob Picker: Blob templates used to pick particles are flipped (dark-on-light to light-on-dark)

  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. 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. Topaz Train: Preprocessed micrographs are inverted (light-on-dark to dark-on-light) before being used for model training

  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.

Phase Plate Workflow

Negative Stain Phase Plate 4

Use the "Phase Plate Data" toggle in Import Movies or Import Micrographs to do a phase shift search during CTF Refinement

Jobs affected by Phase Plate Data

  1. Import Movies: "Phase Plate Data" toggle will set mscope_params/phase_plate to 1 (True)

  2. Import Micrographs: "Phase Plate Data" toggle will set mscope_params/phase_plate to 1 (True)

  3. Patch CTF Estimation: Enables phase-shift search & refinement

  4. CTFFIND4 Wrapper: Enables phase-shift search & refinement

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