Tutorial: Negative Stain Data

Learn how to work with negative stain data in CryoSPARC.

Marking data as negative stain data

Negative stain data must be treated differently from cryo-EM data in several important ways, summarized below. CryoSPARC will make these adjustments automatically when the data has been marked as negative stain data, either during import or later on using Exposure Tools.

Jobs and parameters affected by negative stain data

Job Type	Difference
Import Movies	"Negative Stain" toggle will set mscope_params/neg_stain field to 1 (True).
Import Micrographs	"Negative Stain" toggle will set mscope_params/neg_stain field to 1 (True).
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.
Exposure Tools	Allows user to set mscope_params/neg_stain and mscope_params/phase_plate manually.
Manual Picker	Extracted particles are indicated with the correct sign (+1 for dark-on-light particles and -1 for light-on-dark particles).
Template Picker	Templates used to pick particles are flipped (dark-on-light to light-on-dark).
Blob Picker	Blob templates used to pick particles are flipped (dark-on-light to light-on-dark).
Local Motion Correction	Extracted particles are indicated with the correct sign (+1 for dark-on-light particles and -1 for light-on-dark particles).
Extract From Micrographs	Extracted particles are indicated with the correct sign (+1 for dark-on-light particles and -1 for light-on-dark particles).
Topaz Train	Preprocessed micrographs are inverted (light-on-dark to dark-on-light) before being used for model training
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.

Indicating negative stain data during import

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).

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).

Marking an existing set of exposures as negative stain data

Use the Exposure Tools job to manually set the Negative Stain 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:

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.

Picking negative stain data

In some cases, CryoSPARC's blob picker may not pick the highest-contrast particles in a negative stain dataset. If this occurs, picking may improve if the particle diameter is set to a lower value.

The high contrast at the edges of a negative stain particle creates a significant amount of contrast. This makes the power score slider of the Inspect Particle Picks job particularly effective at selecting these particles.