Wednesday 04/23/14

8:30a – 10:45p

Career planning

  • from Jeff: K99 next application deadline is June 12th. After that it’s October 12th (see NLM page, NIGMS not as clearly laid out).
  • recommended to apply with time to submit resubmission ~8 months after original application. Application and any resubmissions must be received before the applicant starts the 4th year of post-doctoral training. This un-necessarily ambiguous terminology is actually strictly interpreted as 4 years from the date on your PhD, it doesn’t seem to matter what you’ve been doing with the exception of clinical training or parental leave. See FAQ
  • This means to have a hope of being able to submit a revision in response to reviewer feedback, I need to apply in June.

Project 2

The little tasks

  • copying data off temp-drive on STORM2
  • keeps on failing / interrupting due to system restarts in attempt to get lasers and fluid controls to both communicate properly with computer.
  • cancelled data copying. With the temp-drive now recording data and the target drive analyzing data copying more data from temp to target is impossibly slow and it good Windows fashion makes the analysis impossibly slow as well.
  • CopyOnArrival functions in matlab-functions don’t copy folders, just files. Would be good to fix this.

Chromatin Project

Data Processing catch up

  • calibrating DaoSTORM parameters for second round L3E01, L3E07, L2F7F6, L2F7F6 pt 2
  • running DaoSTORM fitting on second round L3E01, L3E07, L2F7F6, L2F7F6 pt 2
  • haven’t been good about getting regular chromatic and z-calibration beads for recent data sets. Need to get better.
  • running analysis of STORM2 beads from 03/28 data set to apply to L3C01-C08 data for the runs taken on STORM2.

Data Analysis

  • lots of interesting big spots from L3C01 (250 kb Y) domain
  • Processed 88 spots, up through C01_storm_0_30.

New coding

  • current CalcChromeWarp is a little bulky, doesn’t do dual view.
  • rewriting CalcChromeWarp from scratch
  • Using region finding instead of splitdax to speed things up a bit. This has a few new challenges:
    • insightM overwrites filenames, since filenames have to match daxnames and all daxnames are the same
    • insightM gives pop-up warnings (InsightM crashed) when movies are blank (e.g. no 488 data). This is annoying.
    • solve this by switching to single iteration DaoSTORM
    • ROI is going to have offsets — beads indexed to their positions in the original file. Will need correct these offsets.
    • fortunately I save mlist_pars, should be able to correct.
  • substantial changes to RunDotFinder to be able to pass alternate binnames. Maybe want to switch back to development branch for regular analysis until we are ready to take this further.

Teaching

  • getting Bogdan trained on STORM3
  • set up daxwriter branch on STORM3
  • blead-through beads look alright for STORM3 without dual view.
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Tuesday 04/22/14

9:30a – 8:30p

issues with z-calibration

  • fixed bug missing sqrt() in wx = w./sqrt(ax) and wx = w./sqrt(ax)
  • take zcal bead images. Run Z-calibration script on these to generate curve fits
  • use the curve fits to refit the beads, plot the reported zs. Should be by design mapping to 0, jump, linear ramp -600 to 600, drop to 0.
  • This is not what we observe: still doesn’t have the correct z-range. Off by a factor of 2.

calCurveTroubleshooting

zcalOffBy2

Microscope meeting

  • add dual view to STORM3 (me?)

issues with 750 STORM

  • yesterday, detected no 750 staining on sample G6-G8 previously imaged beautifully in 750 over the weekend (when the 647 laser was off!)
  • today tried probe hot wash in water-bath at 60C on heat block pre-heated to 60C. Measured temperature in deep filled well also reached 60C. Sample may have been up to ~65C.
  • no 750 spots detected, just very few stray dyes switching.
  • looked at imaged F7-647,F6-750 samples, also detect no staining in 750. Previously observed great 750 spots on this very sample imaged on the 16th and 17th.
  • however do detect decent staining still on the F6-750,F7-647 sample not imaged before, even in BME.
  • hypothesis: the dyes pop off.

STORM

  • imaging F07 (En +2 TAD)
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Monday 04/21/14

9:00a – 6:00p, 10:00p – 12:00a

Lab meeting

  • see notes
  • post-doc candidate interview
  • individual post-doc candidate meetings

STORM

  • attempted to re-image G08-P3-750 G06-P1-647 sample. All 750 spots have vanished! buffer issue? poor secondary stickiness issue?
  • Imaged L3 E08 samples using L3 E09 parameters
  • setting up overnight imaging of E10 samples

Computer craziness

  • Monet still doing mouse stuck to side of screen
  • Turns out to be caused by Dell monitor USB attachment. Plugging in the monitor the system now says ‘installing drivers’ then once this is complete sticks the mouse to the side of the monitor screen, from which it is very hard to dislodge and to which it immediately returns.

Presentation prep

  • working on slides for Penn talk
  • shared slides with XZ for comments

New cell stains

  • L3F7-P1 (1.2 uL)
  • L3F8-P1 (1.5 uL)
  • L2 G08-P3 + G06-P1 rpt (4 uL)
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Thursday 04/17/14

STORM

  • prism2 lost focus just after midnight (~minutes after I checked it and went home)
  • prism2 now upgraded to STORM1. Jeff looking into peizo replacement. (How about a piezo swap with STORM4 dual-objective?)
  • continue imaging on STORM2 sample E07

Staining

  • forgot to check oven calendar, need to wait until 2pm to wash out probes.
  • washing out probes on 6 new samples (3 dual-color lib2 samples and L3 E8 to E10 single color samples)
  • Jeff recommends trying D2O imaging for low quantum yield dyes (e.g. A750).

Modeling

  • test: expected polymer mixing.
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Wednesday 04/16/14

9:00a – 11:45p

Goals

  • project 2 team meeting
  • project 2 design new experiments
  • start analysis

Project 2

General notes

  • wordpress site / notebook running very slow today.

STORM

  • imaged beads
  • imaging E07 on prism2

New hybes

  1. ~4/11 cells (RNase treated), G06-p1 + G08-p3
  2. ~4/11 cells (RNase treated), F07-p1 + F06-p3
  3. ~4/11 cells (RNase treated), F06-p1 + F07-p3
  4. 4/9 cells, newly RNase treated L3E08
  5. 4/9 cells, newly RNase treated L3E09
  6. 4/9 cells, newly RNase treated L3E10

Thicker glass, lower temperature (coverglass vs. microscope slide)
TempCheckGlass

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Tuesday 04/15/14

9:30a – 11:55p

Computer stuff

  • troubleshooting Cajal
  • 590 GTX graphics card fried
  • installed AMD card that came with Dell
  • not recognizing all RAM (only 40 of 48 GB recognized)
  • Turned off, pressed all RAM firmly into DIMM slots. Now only 24 GB recognized.
  • enough of this, this is up and running again, will play with this another time.
  • Quadro5000 for less than $1000: http://www.nvidia.com/object/buy_now_results_ci.html?id=QD5000

working on automatic launching

  • trying to get Matlab to drag-and-drop open files.
  • This seems not to be a solved problem, except for some suggestions of writing stuff in java or using the activeX controls, neither of which I find clear evidence of successful implementation.
  • However one should be able to configure specific filetypes to launch specific matlab functions according to this.
  • Windows 7 no longer has a file type manager window. This program allows access to this functionality: http://defaultprogramseditor.com/download
  • didn’t work.
  • can create a .bat program probably that takes the filetype as its import and calls matlab -r LaunchSTORMfinder(‘daxname’) to window system. This will launch a new version of matlab and open the file. Still not really what we wanted.

Seminar

Probe troubleshooting

  • 3 degree change seems to have made substantial difference in labeling efficiency.
  • sample C01 at 78 thermometer temp denature looks much much better.
  • sample E06 looks nicely stained.

STORM

  • imaging sample E06 on STORM1. Lots of switching.
  • buffer 2 uL COT, 5 uL BME otherwise standard
  • aligned dualview
  • took bead field images and bead Z-calibration images first.
  • Blue-screen system failure. started defrag. deleted some old data files. cancelled defrag (taking forever) Restarted analysis, writing of ESATA this time
  • Blue-screen system failure 2. deleted more data files that users confirmed are backed up. ran windows security updates. restarted defrag. Cancelled defrag (16%). Started analysis. Saving to c/users/data folder (oops). Set partway through movie to have working dir as external ESATA. We’ll see if it switches.

project 2

  • working on slides for XZ for meeting on Thursday. Need slides for Wed. (see googledocs presentation)
  • working on data analysis code.
  • see notes.
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