Confocal images of live and fixed cells with different fixation methods
Using automated image processing to ID effects on chromatin structure.
MEOH fixed sample
Experiment vs Negative control
Switched from simple rescaling to feature finding using FIJI / MIJI
11:00 am – 3:30 pm, 6:30 pm – 11:15 pm
- STORM2 froze during the night (click error provided an out of frame crash in python — built in check doesn’t catch it.
- debugged this with Bogdan, changed catch statement to check size of the object queried, not just the expected size of the image.
- restarted imaging of Ph-KD L2F03 with fresh buffer
- acquisition still looks good at movie 18. And rolling.
- need to take z-beads and chromatin beads tomorrow before Colenso starts.
- need to design and order new Green probes.
- need to analyze existing green data.
- figuring out parsing of new data schema
- trying to process L12 two-color data for analysis with Pipeline 5 or updated pipeline 1.
Nuclei live imaging
- took time lapse images of Hoechst stained nulcei (2 year+ expired DRAQ5 didn’t look that good at 1:10,000)
- took images of mock fix (replaced growth media with more growth media)
- took images of formaldhyde fixed cells
- took images of MeOH fixed cells (clearly shrunk)
- need to do some coding to analyze these.
- probably worth some manual playing with the Fiji tools too.
- some issues with Z-focus and x-y registration, largely sorted though.
- check that numbers don’t change much upon down-sampling
- reading article, drafting comments of first impressions
- very long methods section, reading imaging methods in detail
11:00 am – 7:30 pm, 9:30 pm – 10:45 pm
(9:00 am – 11:00 am volleyball practice)
- plated Kc167 cells (in SFX) on glass
- my more recent flask with the short Trypsin treatment is doing well.
- the original passage with the longer Trypsin exposure is not looking so good.
- tried removing Psc- cells by blowing (without Trypsin). works a little bit, not at all like with Trypsin.
- added a little scraping an moved cells to new vial
- tried imaging cells live labeled with Hoechst – doesn’t bleach much or blink
- tried imaging cells live labeled with Draq5 – doesn’t bleach at all (or switch to dark state to blink)
- tried both culture media and STORM BME imaging buffer.
- Invitrogen has a number of other cell-permiable dyes: https://www.lifetechnologies.com/us/en/home/references/molecular-probes-the-handbook/probes-for-organelles/nuclear-and-chromosome-counterstaining-and-nissl-stains.html#head1
Cell cycle markers
- CycA (DSHB)
- phospho-H3 (M-phase specific) see here (fig 1, red)
Culturing Psc- cells
- tried passage by scrapping — disaester, cells die
- tried trypsin first spin looks good. No cells came down in PBS spin (sparse cells). Don’t look good on plates. I think I killed these.
9:10 am – 8:00 pm, 9:30 pm – 12:00 am
- confocal imaging
- design primers
- re-read and submit review
- confocal imaging (no staining)
- submitted review (done)
- feedback for Hao on essay (sent)
- no signal with 37C hybe
- to try next: 10% formamide, 2X SCC, 10% dextran sulfate, 37C
Working on Ph co-localization data
- see notes
- sent updates to NJ and AW
- analyzing Ph-M co-colocalization Flag Ph
- analyzing conventional data
- discussed approach
- to start MERFISH experiments in Drosophila embryos
- Plan: use Biosearch probes to optimize RNA labeling using DNA probes
- currently using 20mers directly labeled. These aren’t working.
Images from Shawn Little’s paper (Gregor lab)
My RNA hb stains
zoom out on whole embryo, stained in green for hb
a different embryo with hb in red and an intronic reporter for a hb transgene (at a different insertion site on a different chromosome) in green
My DNA hb stains don’t work at all (following protocol from the Little et al Cell paper)
- Not clear why
- could repeat RNA stains again just to make sure everything is working alright. Not sure if my RNA-probes are still good after 6 years in the -20C but I can try.
- try different hybe conditions (tried RT hybe, which is what appears to be what Little et al use, tried 37C hybe).
- could try different hybe buffers
- I recall Raj reported not being able to get Drosophila embryos to work with his 2008 protocol
9:30 am – 7:30 pm, 9:30 pm – 11:45 pm
- reviews back
- writing reply
- goal to respond this week
- Ph-Flag / Ph co-localization data
- DNA FISH long range contact frequency (hold off)
Live imaging of chromatin
- dCAS9 for Drosophila sequence
- transfection vector for Drosophila
- dCAS9-fused to Fok1 (need to add mmaple3) from addgene (here)
- repeat sequences:
- the “dodeca repeat” ACGGGACCAGTACGG
- the “359 repeat” GGGATCGTTAGCACTGGTAATTAGCTGC,
- the “AACAC” repeat: AACACAACACAACACAACACAACACAACACAACAC
- the AATAT repeat: AATATAATATAATATAATATAATATAATAT