[CryoEM] high resolution 2D class images from cryoSPARC

2D classification is an important step to identify good particles/2D groups for further 3D structural classification and following calculation. In RELION (begins at 3.0, I guess), we can use mouse right button to save PNG images of individual 2D averages. In cryoSPARC, there is no such function provided yet (I have v3.2 now). cryoSPARC does provide nice tiled image(s) of 2D averages after calculation, however, there are labels of group, resolution, particle numbers and other information. It’s not trivial to get a “clean” image of 2D average.

I found a cryoSPARC forum discussion related to generation of high resolution images of each 2D average using cryoSPARC result. There are few solutions, including (1) python scripts provided by cryoSPARC, (2) EMAN2 scripts and (3) combination of Fiji and image editor. I personally found the 3rd option is easier to handle and can be fully operated in my macbook without using additional cryoEM scripts.

Here are the procedures I used to get tiled 2D average images. I use a 1.86 Å map of mouse apoferritin calculated by cryoSPARC 2.14 as an example in this post. The 2D classification averages are beautiful and there are several good 2D views I want to use as figures for paper, presentation or poster. The green labels are not easy to be erased and I will show steps by steps to make my own tiles of 2D views.

First, download the “2D average” mrc file clicking the “2D averages” panel in the job (See figure below). The filename usually looks like “cryosparc_P5_J18_020_class_averages.mrc”

Then get “Fiji” installed. Fiji is modified from ImageJ and can read MRC files without problem. In my macbook, I use Fiji to open the cryosparc_P5_J18_020_class_averages.mrc file and the open window has a scrollbar at the bottom representing numbers of frames in this mrc. The frames are individual 2D views. The total frame number can be found at the top-left corner of this class_averages.mrc window in Fiji.

The default image size is 64 x 64 with 25400 dpi. There are two ways to change the image to larger size. Then export individual 2D views for manual arrangement in poster or figures. One is done by Fiji itself and the other one is using Preview in Mac.

For the Fiji way, 
(1) go to toolbar, select "Image", choose "Adjust", then pick "Size", then users can adjust the size from 64x64 to larger number.  I often change it to 256x256.

(2) we go to "Image" --> Adjust --> Brightness/Contrast to adjust the background darkness.

(3) For non-mac user, a simpler way is to export all "stacked images" to individual ones.
Image --> Stacks --> "Stack to Images"

(4) manually delete unwanted 2D views and save the individual good 2D views to JPEG, PNG or whatever image format you like to use for.

Below has the tiled 25 good 2D views I selected in Fiji, then tile them together in powerpoint.

The "preview" way is a bit different.
I like to use Preview of MacOS and the Preview does provide ways to handle these stacked tif images. Before using Preview, we need use Fiji to "export" the multi-frame 2D views as "tif" file first.  Then open the tif file (named 1.tif) by Preview of Mac.  

Tools --> Adjust size, give the new number you want. 

I also often deleted unwanted 2D views at this step. To export one frame by one frame, I found that I must save the edited 1.tif file to new TIF image (2.tif at here). Then open the new 2.tif and save one frame by one frame using "print" or simply drag the thumbnail to folder, Mac will generate the image for you. Simply repeat several times to get all 2D views saved.

Below has 3 2D views tiled together after I dragged each frame to a folder. The default contrast is high and we can use any photo editor to alter the brightness, contrast and other graphic parameters to present a dark gray background like what cryoSPARC did.