Import LZW-compressed TIFF from Falcon 4/EPU 3.2

Import and Export
1

I haven’t found this issue addressed here - using EPU 3.2 with a Falcon 4i camera, I have been saving movies in the newly available LZW-compressed-TIFF format that was previously possible only for K2/K3 cameras. However, while I can read these movie files into RELION4, I can’t import them into cryosparc 4.0.3:

Importing movies from /nethomes/home1/conway/executor/2022-12-01_TestResolution/1-Standard-AFIS/relion4/Movies/*.tiff
Importing 1701 files
Import paths were unique at level -1
Importing 1701 files
Reading header for each exposure…
====== Job process terminated abnormally.

I have tried the following:

  • checked permissions along the whole path
  • used “Skip Header Check” but it still checks the first header
  • selected different small subsets of files (so there isn’t a single bad file, they are all bad)

There is no gain reference as the Falcon 4i already accounts for it in the movie fractions. Other parameters are unremarkable:

  • Raw pixel size 0.415 A (these tiffs seem to be super-resolution, 8k)
  • accelerating voltage (kV) 300
  • Spherical aberration (mm) 2.7
  • Total exposure dose (e/A^2) 50

Unfortunately, cryosparc offers no information to pin-point what it can’t handle with these files - is it the header, the path (no special characters, no spaces), something else? My first question is: are these Falcon/EPU-generated compressed tiffs compatible with cryosparc? If they are, has anyone successfully imported such files? Am I missing some step to import them?

Thanks for any pointers,

James Conway

2

Are there any error messages in job.log inside that job’s directory?

4

(Sorry, a return key got away from me)
There is nothing useful in the job.log file:

================= CRYOSPARCW ======= 2023-01-04 10:51:44.353417 =========
Project P2 Job J12
Master a.b.pitt.edu Port xxxxxx (obscured)

========= monitor process now starting main process
MAINPROCESS PID 37589
========= monitor process now waiting for main process
MAIN PID 37589
imports.run cryosparc_compute.jobs.jobregister
========= main process now complete.
========= monitor process now complete.

5

PS. I can send a link to an example tiff file, if this is helpful. Compressed movie file size is 278 MBytes. There are 34 frames (fractions) and the size is 8k x 8k, 8-bit (2.1 GBytes uncompressed).

6

This would be helpful indeed. Please send me the link by direct message.
If you used a gain reference and/or defect file during import, please include that information also.
Thanks.

7

Thanks for following up. Link sent, no gain reference or defect files used, just importing the movie files.

8

Is there any diagnosis of what the issue is with these TIFF files? I am stuck with this. I also note that the Falcon 4i non-movie TIFFs (not compressed) also don’t read into cryoSPARC and seem to have a similar error. Any update appreciated.

9

Thank you for providing the sample movie, but we have not yet examined the issue.

10

Hi @jxc100,

We’ve had a chance to look at the tiff file you uploaded, and this is indeed a cryosparc bug. I will update you when a fix is available.

—Harris

11

Hi @jxc100, this issue has been fixed in the recently released CryoSPARC v4.1.2. Thanks again for bringing it to our attention.

12

Sorry, but I have another issue with importing EPU/Falcon TIFF files that I think is related to the one fixed. If I disable “Skip Header Check” then the import fails after some variable number of movie files (usually around 90, but sometimes as many as 200 - so it’s not one bad file) with:

Job is unresponsive - no heartbeat received in 60 seconds.

If I check “Skip Header Check” then the import of ~2000- movies complete. I see that cryosparc (4.1.2) reports “Unknown field with tag 65001 (0xfde9) encountered” for each frame, but this shouldn’t be a problem as its just a tag defined by FEI and not important for cryosparc. IA job.log file with the failure is at this link:
job.log

Thanks for any help.

James Conway