Monday 01/27/14

9:45a – 11:55p

Meetings

  • Group meeting, Jeff presents (see protected notes)
  • Journal club (see notes)
  • Meeting with Ajaz, get antibody
  • Meeting with Brian B, discuss figure
    • put chromatin loop image last as speculative difference
    • for main figure, combine replicates in cdf plots. Put KS tests on plots
    • Also combine replicates in bar plots, put Wilcoxon/Mann-Whitney stats on plots
    • open with a couple images of each region just showing photon differences.
    • all background levels analysis move to supplement
    • replicates as separate bars and curves, move to supplement.

ChromatinCropper Analysis

  • z-warp map needs to become a parameter that gets saved with the data
  • analyzing more black chromatin data. basically finished black series.
    • F01 has pretty high background and should be repeated. I tried several analyses to see if I could get the background to intefere less with the volume measurements.
    • F10 has pretty low signal density and should be repeated.
  • see snapshots of progress
Posted in Summaries | Comments Off on Monday 01/27/14

Journal Club 01/27/14

Doory

Ultrafast endocytosis at mouse hippocampal synapses
Nature 504, 2013

Background

  • lab previously published correlative EM
  • Combine optogentics and high-pressure freezing ‘flash and freeze’, to study synapse.
  • 40 nm EM sections
  • methods of exocytosis
    • full fusion (vesicle fuses and releases)
    • kiss and run – transient fusion, partial release of cargo, partial transfer of membrane?, vesicle still exists
  • methods of endocytosis
    • clathrin mediated
    • ‘kiss and run’ – transient fusion reverses, (some membrane transfered to vesicle? some cargo endocytosed from outside during transient fusion?)

Observation

  • Docked vesicles fuse in the active zone.
  • estimate number of vesicles exchanged per synapse per stimulation (?)
  • Endocytosis peaks at 100 ms (judged by size and by incorporation of a fluid label).
  • exocytosis in the active zone, endocytosis at the periphery.
  • show that actin is requried from endocytosis.
  • Dynamin is required for proper cleavage of endcytosis events.
  • no evidence of intermediate size vesicles (kiss and run).

Main conclusion

  • endocytosis recycling at synapses is occurring much faster than previously believed (100 ms)
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Protected: group meeting 01/27/14

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Sunday 01/26/14

10:20a – 10:00p

Literature

Chormatin Project

Data Analysis

  • add chromatinData folder as a global to Matlab startup.m.
  • not all chromatin plots have been analyzed with the same z-data.
  • Rewrite 3D analysis of CC to use z-calibration curve.
    • note: DaoSTORM does Z-fit as part of dot fit when using Z method. We are now doing a post-analysis z-fit. These behaviors seem to give rather different values unfortunately (or maybe all DaoSTORMs fits give different values, I haven’t actually re-run it in 3D mode instead of Z mode).
  • continuing analyzing blue regions. Plot comparative results as we go.
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Saturday 01/25/13

10:30a – 5:30p, 7:00p – 8:00p
remotey 9:30p – 11:45p

Chromatin Project

Data analysis / code development: ChromatinCropper

Integrating 3D images

  • working on 3D image masks.
  • Fixed up Stats2DScatter
  • currently hard-coded in z-range is -300 300 corrected x4. Our calibration curves are substantially off. Should stick some small beads on cells sometime soon and recompute these curves.
  • should filter out spots and recall the list2img, not create binary masks.
  • need to export area masks for XZ as well.

New functions

  • MaskMoleculeList

Start data analysis

  • Analyzed new BXC data
  • Analyzed G9 data
    • has some big spots as well as small ones, and some substantial variation
    • some of this may be due to pairing
    • maybe larger polycomb domains hold better compact.
    • propose an additional evolutionary pressure to cluster the genes.
Posted in Summaries | Comments Off on Saturday 01/25/13

Thursday 01/23/14

9:45a – 12:15a

STORM analysis

  • analyze bead data for BXC double stains
  • analyze bead data for PhM Psc
  • Finish O/N run of G05
  • quick look at ANTC data (just image based drift correction available at present)
    • fewer localizations despite bright dyes with MEA. This is expected
    • Many clusters not too compact. Radial symmetry of G09/En/BXC generally less evident

ChromatinCropper

Redesigning data storage structures.

  1. Each saved dot is its own object with its own data
  2. Each folder is also keeping a structure that has a running record of all the data for all the spots.
  3. Each spot should get processed individually. We want to look at these one by one and decide whether it’s sufficiently in frame, properly thresholded etc. Not do it in batch and ask if the batch all looks good. This requires a dramatic redesign of step 6 and 7.

Recording spots

  • spot per image leaves odd blanks and turned out to be hard to trace back.
  • each spot is just going to have all its own image data and all its own meta data saved directly with it.
  • just insert save dot in linear array
  • potential issue: if you click save dots twice, the data will get double entered.

Step 6: Filter Dots

  • Let’s use the dot slider as the advance dot.
  • Add a new button to save the dot after examining it. None of this apply a bunch of stuff to all the dots in this image and then chose which ones are good enough to save. Hopefully saving dot by dot also reduces the chance of double entry. It’s no longer a ‘step’ that I might be I’m not sure if I ran it or not. Well, that, and hopefully the printouts to the

Data structures

  • large cell arrays of structures not a good format. Hard to index out of.
  • i.e. don’t use dataobject{i}.datatype use dataobject.datatype{i}

New General Purpose functions of interest written today

  • structOut = IndexStructure(structIn,idx) For structures full of cells and arrays that are all same length (e.g. mlists) and you want to index out of them.
  • clrmap = GetColorMap(colormapName,pts) Returns more colormaps (including all my new black to white via blue, black to white via green etc ones).
  • [stormXZ, stormYZ, stormXY] = List2ImgXYZ(mlist) Generate XZ and YZ projects from 3D data

3Dprojections_AxisImage

Of interest

git log -p --follow filename the --follow will track the file through moves within the repo and file renaming.

Feedback

  • read Carl’s proposal and provide feedback.
  • discuss proposal with Carl.
  • Still to do!: reread latest version of Hao’s write-up and provide feedback.
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Wednesday 01/22/14

9:30a – 11:50p

Chromatin project

Planning

  • write up summary of yesterday’s meeting with XZ
  • work on improving metrics for comparison of chromatin regions
  • writing up plans for new ChromatinCropper to implement revised analysis
  • plans for sequencing validation
    • up to 12 lanes per library can be fully double indexed and distinguished (and 96 total lanes run in parallel on MiSeq)
    • 3 finalized probes, 2 fully independent replicates. (up to 6)
    • 2 independent PCRs of all 40 Drosophila loci (can run one 1 plate).
    • 2 limited cycle PCRs of 2 loci, (run on QPCR machine)

STORM

  • imaging G05. I still think these domains are too large. It will be very interesting to see how they compare to the rest of ANTC.
  • Analyzing ANTC data. Forgot to use my nice new daxwriter bead integration. Going to have to fit the beads separately.

Secondary Projects

Project 2

  • revise writeup for Hao / XZ.

Ph Project

  • image / test all new antibody stains. Find which 750 range dye looks the best
  • m Alexa-750 #46 gives the best stains. I’m suspicious the bright dots one per cell here due to the secondary. But the rest of nuclear staining looks good. We’ll see if these dots cluster etc.
  • Some of these dyes give no staining at all. Or maybe some cytoplasmic background but no
Posted in Summaries | Comments Off on Wednesday 01/22/14

Chromatin Project, short-term goals

Issues with current data analysis

  • In trying to get of order 100 loci per chromatin domain I’ve take a bunch of partially imaged edge of focus domains or other less well defined images (e.g. near edge of field of view). These un-necessarily blur out the size distributions of regions. A more stringent filter will be better.
  • I think this is maybe even more important for statistics like moment of inertia and total localizations
  • I haven’t used the z-scan information for number of dots per cell. This is pretty important. We should make this a field of the export data so that each spot has

Changes to make moving forward

Better Data Filtering

  • Let’s get Z information and other quality metrics into the chromatin cropper.
  • Let’s plot the spots statistics on a scatter plot so we can see where each of them go

Better Data storage

  • the two to three step annotation process is unnecessarily slow. This should all be done in the Chromatin Cropper
  • Each individual dot should save
    • its raw data
      • vlist
      • imaxes
      • impars
      • imdata (Iconv, Istorm, Itime, Ihist XY YZ XZ, Icell, Iarea). For just that dot
    • its ID information
      • region ID (e.g. F03). This can be used to retrieve locus size, locus coordinates, ChIP seq details etc.
      • daxfolder
      • binname
    • its dot quantification statistics
      • Area
      • Area Map
      • moment of inertia
      • something like distance of local maxima from edge. Distance of internal local minima from edge. Define maxima/minima based on quantiles. Say local max are all the spots that hit 90% or above. Local min are all the local min that remain if we saturate 10% max and above.
      • Coefficient of variation for localizations per pixel
      • Water-filled areas
    • png images
      • use export_fig.m function with set(fighandle,'PaperPosition',[0,0,width,height]). This will capture the set(gcf,'color','k') and other properties, and preserve the spacing.
      • conventional and STORM dot pair. Just re-apply an auto-contrast for the conventional image and then convert to the correct colormap. STORM image may require manual contrast adjustment
      • Area

Experiment planning

Imaging Priorities

  • G05 647
  • D12 647

Staining Priorities

  • F03 + F04
  • F05 + F04
  • G01 + G02
  • G01 + G02 + G03
  • G01 + G02 + G03 + G04
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Protected: Library2 Data Summaries

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RT smears troubleshooting

A quick look at my history of RT success to ID what started going wrong?

Observations

  • not much smearing in early samples. Incorporation rate increases with total amount of mRNA added.
  • Using beads to concentrate mRNA increases production.
  • Using pure mRNA T7 further increases production
  • Small samples have worse smears just after the high quality maxima integration (8 probes at 800 pmol P1, and 1 at nmol P2, 2 at 600 and 800 pmol P2).

The decline

  • Correlates with switch to new P1. P3 was doing better than P1 for a little while after the switch.
  • correlates with switch to murine RNase inhibitor from NEB (cheaper). Best runs were with RNasin Plus.
  • This is interesting, RNasin plus advertises thermal stability up to 70C. Maybe this is the trick — NEB’s inhibitor says it should be used below 50C.
  • correlates with lower IVT yields
  • Same protocol essentially. Had higher maxima when we got high incorporation, but bringing maxima back up to high concentration doesn’t restore high incorporation.
  • Nothing else changed really in protocol.

IssuesWithRTs

Ideas

  • some contamination of reagents.
  • switch of RNase inhibitor.
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